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Lecture 1
Lecture 1
Central Dogma and Deciphering the Genetic Code
Hartmut "Hudel" Luecke
Department of Molecular Biology and Biochemistry
Area of reseach: Structure & Function of Proteins
Email: [email protected]
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Lecture 1
http://bass.bio.uci.edu/~hudel/
bs99a
http://bass.bio.uci.edu/~hudel/
bs99b
Next: The Central Dogma
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The Central Dogma (1.1)
The Central Dogma
DNA
4 nucleotides (A,C,G,
T)
==>
RNA
4 nucleotides (A,C,G,
U)
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==>
Protein
20 amino acids
The Central Dogma (1.1)
TGGCGAACTGATGTG
transcription
by
UGGCGAACUGAUGUG
polymerase
phosphodiester bond
translation
by
TrpArgThrAspVal
ribosome
phosphodiester bond
peptide bond
Comparing proteins with nucleic acids:
Properties proteins have in common with nucleic acid:
●
●
Linear heteropolymers with a defined sequence
Individual building blocks (called amino acids or simply residues for proteins) are linked
together through covalent (chemical) bonds
Properties different from nucleic acid:
●
●
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More diverse building blocks: 20 amino acids vs. 4 nucleic acids
Large variety of functional groups: negatively charged, positively charged, hydrophobic,
hydroxyl, sulfhydryl
Vastly accelerate a multitude of chemical reactions (also: ribozymes)
Assume a wealth of well-defined tertiary structures (shapes): helix bundles, beta sheets, alpha/
beta barrels etc.
What do proteins do?
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●
●
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Catalyze chemical reactions (enzymes): alcohol dehydrogenase
Carry nutrients: hemoglobin is the oxygen carrier in your blood
Signaling: peptide hormones bind to protein receptors, transcription factors
Molecular recognition: antibodies bind to antigens
Play structural roles: finger nails, hair, eye lens
Function as motors & pumps: myosin-actin in your muscles, ion pumps
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The Central Dogma (1.1)
Proteins are the molecular workhorse of the
cell
Proteins are of central importance in every
cellular process
Proteins must be made by the cell with high
fidelity
Examples of single amino-acid mutations that cause disease:
●
●
●
hemoglobin: Glutamic acid (Glu) to valine (Val) mutation at position 6 of the beta chain
causes sickle-cell anemia.
Fibroblast Growth Factor (FGF) receptor 3: Glycine (Gly) to arginine (Arg) mutation results
in achondroplasia, a form of dwarfism for which the gene was discovered here at UCI by Drs.
Thompson and Wasmuth.
Enzyme uroporphyrinogen III cosynthase: causes congenital porphyria with symptoms such
as skin photosensitivity & scarring, mutilating skin deformity, hypertrichosis, hemolytic
anemia, red stained teeth.
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The Central Dogma (1.1)
Next: Translation
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3.4 Primary through Quartenary structure
Primary Through Quartenary Protein Structure
This is a molecule of hexokinase, a metabolic protein found in almost all living organisms. This protein
is composed of approximately 6000 atoms and weighs 48 kiloDaltons (kDA). It also has a glucose
molecule bound to it, but that is nearly impossible to see.
Primary Structure
Protein have multiple levels of structure. The most basic is its primary structure. A protein's primary
structure is simply the order of its amino acids. Note that this order is always written from amino end to
carboxyl end (by convention). An example of a protein primary structure is:
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3.4 Primary through Quartenary structure
1
31
61
91
121
151
181
211
241
271
301
331
361
391
421
451
A
T
G
S
X
T
X
S
V
A
X
L
I
G
K
X
A
T
S
R
E
X
X
G
C
X
H
R
A
S
P
X
S
R
F
S
F
Q
V
V
C
K
X
X
A
X
I
S
X
X
L
L
S
A
A
N
X
T
X
X
Y
R
X
A
5
D
D
A
A
S
X
D
A
Q
F
X
X
A
D
I
X
X
D
I
A
X
A
I
A
D
E
A
D
F
Y
T
X
S
X
V
S
A
F
X
Y
S
K
X
L
R
S
P
A
L
D
M
M
G
S
D
W
F
N
D
F
L
G
A
10
V E
S A
G G
X T
S V
L A
S H
C D
R K
S X
F X
X G
V V
F S
I D
V
A
G
T
P
X
G
S
A
A
A
D
C
X
G
H
A
D
A
L
L
I
T
F
K
A
Q
X
N
E
X
S
L
I
G
X
L
X
P
N
N
G
I
S
G
15
X V
I P
E V
P S
F T
K L
X X
I A
S L
X G
V E
I A
X A
A T
A A
F
M
I
D
F
I
V
D
P
Q
N
X
I
X
X
I
V
L
L
X
S
N
A
Q
S
S
K
C
N
X
V
P
I
W
E
A
Y
A
I
L
S
T
Q
X
V
20
P P
G W
X L
G N
A G
M X
T D
D A
X Y
R D
Y P
X M
K K
N I
I X
X
V
A
X
A
N
A
G
X
V
A
K
G
Y
S
I
L
G
A
K
A
X
X
X
L
K
X
Y
G
I
L
K
Y
X
E
X
I
X
T
M
I
V
S
W
A
25
Q A
Q V
Q E
S N
X V
F P
K M
G G
L N
X Y
Q K
V R
S G
P Q
S S
V
X
S
A
I
A
G
A
X
K
L
R
H
S
Q
V
G
S
A
K
G
I
G
X
X
P
X
I
A
X
S
S
I
F
G
D
I
X
S
X
H
L
A
X
X
30
I A
Q A
X A
S S
Q I
X X
F G
M X
P X
G Q
F D
F L
A X
X S
X A
This is the sequence of hexokinase, yeast hexokinase from the yeast species Saccharomyces cerevisiae
to be specific. To find out more about this protein, jump to the Brookhaven Protein Data Bank 3D
browser and enter hexokinase in the textbox, or SCOP (Structural Classification of Proteins) and use the
PDB reference number 1HKG.
Secondary Structure
Protein secondary structure refers to certain common repeating structures found in proteins. There are
two types of secondary structures: alpha-helix and beta-pleated sheet.
An alpha-helix is a tight helix formed out of the polypeptide chain. The polypeptide main chain makes
up the central structure, and the side chains extend out and away from the helix. The CO group of one
amino acid (n) is hydrogen bonded to the NH group of the amino acid four residues away (n +4). In this
way every CO and NH group of the backbone is hydrogen bonded.
Here are three models of an alpha-helix. The first shows only the alpha-carbon of each amino acid. The
second shows all of the atoms that make up the backbone of the polypeptide. The third shows all of the
hydrogen bonds that hold alpha-helices together.The third, and most complete, model is also shown
here.
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3.4 Primary through Quartenary structure
A-helices are most commonly made up of hydrophobic amino acids, because hydrogen bonds are
generally the strongest attraction possible between such amino acids. a-helices are found in almost all
proteins to various extents. For more information read Stryer's Biochemistry, page 27.
B-pleated sheets are the other type of secondary structure. They can be either parallel or anti-parallel.
Anti-parallel beta-pleated sheets generally look like
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3.4 Primary through Quartenary structure
At the turns they have the
structure:
where amino acid n hydrogen bonded to amino acid (n +3) in a hairpin turn.
There is a special type of molecular model used to highlight protein secondary structure. Follow this link
to view an example. This common type of protein model represents segments of beta-pleated sheets as
ribbon arrows, and it represents a-helices as helical ribbons. The remainder of the polypeptide chain is
referred to as random coil, and is represented as a thin line. Please note that random coil is somewhat of
a misnomer; the protein is definitely highly organized, but the random coil regions do not show any
easily categorized secondary structure components.
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3.4 Primary through Quartenary structure
Tertiary Structure
Tertiary structure is the full 3-dimensional folded structure of the polypeptide chain. There are a number
of examples of tertiary in your textbook, and the hexokinase image used as the icon in this module is a
complete structure.
Quartenary Structure
Quartenary structure is only present if there is more than one polypeptide chain. With multiple
polypeptide chains, quartenary structure is their interconnections and organization. This is an image of
hemoglobin, a protein with four polypeptides-- two alpha-globins, and two beta-globins. The red patches
are heme groups (iron complexes bound to the protein, which bind oxygen).
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3.4 Primary through Quartenary structure
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http://bass.bio.uci.edu/~hudel/bs99a/lecture20/ahelix1.gif
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http://bass.bio.uci.edu/~hudel/bs99a/lecture20/ahelix3.gif
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http://bass.bio.uci.edu/~hudel/bs99a/lecture20/ahelix3.gif
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3 Proteins
Amino Acids
Proteins are very complicated moleucules. With 20 different amino acids that can be arranged in any
order to make a polypeptide of up to thousands of amino acids long, their potential for variety is
extraordinary. This variety allows proteins to function as exquisitely specific enzymes that compose a
cell's metabolism. An E. coli bacterium, one of the most simple biological organisms, has over a 1000
different proteins working at various times to catalyze the necessary reactions to sustain life.
1. Amino Acids Diagram
All amino acids have the same general formula:
The twenty amino acids found in biological systems are:
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3 Proteins
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3 Proteins
All proteins are linear chains composed of these 20 amino acids.
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Hammerhead Crystal Structure
Hammerhead Ribozyme Crystal Structure
An image of the hammerhead ribozyme crystal structure as determined by Pley, Flaherty and McKay
("Three-dimensional structure of a hammerhead ribozyme." Nature (1994), 372: 68-74). The ribozyme
is shown bound to an all-DNA substrate inhibitor (in blue), with the 5' end of the substrate (and the 3' end
of the ribozyme) on the left of the image. The catalytic core of the ribozyme is shown in red, stem II in
purple, and the substrate binding arms of the ribozyme (stems I and III) in green.
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Hammerhead Crystal Structure
Click here for general information on ribozymes
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Translation (1.2)
Translation
The first step in following the blueprint of DNA to
make a protein is transcription which generates an
mRNA copy from the DNA template.
Translation is the 2nd step: It uses the mRNA
template to make the protein polymer. This process
is also called protein synthesis.
The reasons for having two steps instead of one are:
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●
●
●
Amplification: a single copy gene on DNA can be transcribed into many copies of
mRNA
Increased levels of control: regulation of transcription as well as translation
Ability to separate the mechanisms for DNA replication & transcription from
protein synthesis
In eukaryotes: Ability to spatially separate replication & transcription (nucleus)
from protein synthesis (cytoplasm)
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Translation (1.2)
Translation is the process of reading the copy of
genetic information on the mRNA (linear sequence
of 4 different nucleic acids) and translating it into
the proper linear protein sequence of 20 different
amino acids.
This process is performed in one of the most
complex organelles of the cell, the ribosome. In the
ribosome the mRNA sequence (information) is read
and the corresponding polypeptide (protein) is
assembled. The rules for translating the linear
nucleic acid sequence (mRNA) into the linear amino
acid sequence (protein) are called the Genetic Code.
Next: The Genetic Code
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The Genetic Code (1.3)
The Genetic Code
How to encode a 20-letter alphabet (protein) with a 4letter alphabet (DNA)?
1 nucleotide
4
A, C, G, U
4 amino acids
2 nucleotides
4 x 4 = 16
AU, AG, CA, UU etc.
16 amino acids
3 nucleotides
4 x 4 x 4 = 64
AUG, UGC, CGA etc.
64 amino acids
Since two nucleotides are not enough (16), three
nucleotides are needed to code for all 20 amino acids
Thus Watson & Crick proposed that codon triplets code for individual amino
acids.
There are several possibilities how triplets might code for amino acids:
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The Genetic Code (1.3)
To verify that the Code uses triplets and to determine:
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●
●
overlapping vs. non-overlapping code,
punctuated vs. unpunctuated code,
the redundancy of the code (64 triplets for 20 amino acids)
the following experiments were performed in the early 60s:
Next: Experimental Evidence for the Genetic Code
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The Genetic Code (1.3)
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Experimental Evidence (1.4)
Experimental Evidence
Crick & Brenner (1961) showed the effect of successive
deletions of nucleotides in bacteriophage T4 DNA:
ATG CTG CTC TGT GCC GCC
Original sequence
Met Leu Leu Cys Ala Ala
ATG CTC TCT GTG CCG CC.
1 nucleotide deleted
Met Leu Ser Val Pro Pro
ATG CTT CTG TGC CGC C..
2 nucleotides deleted
Met Pro Leu Cys Arg ...
ATG CTC TGT GCC GCC ...
3 nucleotides deleted
Met Leu Cys Ala Ala ...
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Experimental Evidence (1.4)
Deletion of 1 or 2 nucleotides (frame shift mutation)
results in non-functional protein
Deletion of 3 nucleotides results only in deletion of
1 amino acid (but protein could still be
dysfunctional)
Insertion of 3 nucleotides results in insertion of 1
amino acid (not shown)
Change of 1 nucleotide results in either a sense or
silent mutantion or in a missense mutation
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Experimental Evidence (1.4)
Nucleotides are read as triplets without
overlap or punctuation
Next: Deciphering the Genetic Code
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Sequence Mutations
Nucleic Acid Sequence Mutations
Nonsense Mutation
A change in the ribonucleotide base sequence that results in a nonsense (STOP) codon. The
protein will be terminated at that point in the message.
Missense Mutation
This is usually a single substitution mutation and results in one wrong codon and one wrong
amino acid.
Sense or Silent Mutation
This is a single substitution mutation where the change in the DNA base sequence results in a
new codon still coding for the same amino acid (Redundacy of the Genetic Code).
Frame Shift Mutation
When a number of DNA nucleotides not divisible by three is inserted or deleted. This causes a
reading frame shift and all of the codons and all of the amino acids after that mutation are usually
wrong. Frequently one of the wrong codons turns out to be a nonsense codon and the protein is
terminated at that point.
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Deciphering the Genetic Code (1.5)
Deciphering the Genetic Code
Which triplet codon corresponds to
which amino acid?
Nirenberg (1961) added synthetic homo-polynucleotides to
bacterial lysate:
poly U
UUUUUUUUUUUU
Phe-Phe-Phe-Phe
poly A
AAAAAAAAAAAA
Lys-Lys-Lys-Lys
poly C
CCCCCCCCCCCC
Pro-Pro-Pro-Pro
Thus the first codon was determined: UUU codes for
phenylalanine
Alternatively, AAA code for lysine, and CCC for proline.
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Deciphering the Genetic Code (1.5)
Khorana (mid 60s) was able to synthesize triplet repeats such
as (AAG)n:
When AAGAAGAAGAAG was incubated with bacterial lysate containing
ribosomes, tRNAs etc., the following results were obtained:
Reading frame
1
Reading frame
2
Reading frame
3
-AAG-AAGAAG-AAG-
A-AGA-AGAAGA-AG
AA-GAA-GAAGAA-G
-Lys-LysLys-Lys-
-Arg-ArgArg-
-Glu-GluGlu-
Upon translation, a mixture of homo-polypeptides (poly-lysine, polyarginine and poly-glutamic acid) was obtained, according to the 3 possible
reading frames. No hetero-polypeptides were produced, confirming the
absence of overlap and punctuation in the Genetic Code.
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Deciphering the Genetic Code (1.5)
In 1964 Nirenberg & Leder developed a filter-binding assay:
Ribosomes were
incubated with one
radioactively labeled
aminoacyl tRNA* (Trp)
and unlabeled (non-Trp)
tRNAs. Subsequently,
one type of synthetic
RNA trinucleotide (AAG
or CGA or UGG) was
added. When this
solution was washed
over Millipore filters,
only ribosome/tRNA/
mRNA complexes where
tRNA* (Trp) AND
mRNA (UGG) were
complementary
remained on the filter.
Free, noncomplementary tRNAs
and mRNA were washed
off.
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Deciphering the Genetic Code (1.5)
UGG
Trp* tRNA
UUU
UUC
Phe* tRNA
In this fashion, the remaining code pairs were determined.
Next: The Genetic Code
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Table of the Genetic Code (1.6)
Table of the Genetic Code
In 1968
Nirenberg
& Khorana
jointly
were
awarded
the Nobel
Prize for
the
elucidation
of the
Genetic
Code.
Noteworthy observations:
●
Most codons for a given amino acid differ only in the last (third) base
of the triplet (exceptions: Leu, Arg, Ser)
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Table of the Genetic Code (1.6)
●
●
One codon (AUG or Met) also signals the START of a polypeptide
chain.
Three codons (UAA, UAG and UGA) are used to signal the END of a
polypeptide chain (STOP codons)
For a historical account of the cracking of the Genetic Code click here.
Next: Summary
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Summary (1.7)
Summary
Features of the Genetic Code
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The Code transfers information from mRNA to proteins with high
fidelity
It is redundant or degenerate: 61 mRNA triplets code for 20 amino
acids
Contains START (1) and STOP (3) codons
The Genetic Code is nearly universal: correspondence between a
nucleotide triplet and an amino acid is identical from viruses to
mammals. The rare exceptions are mitochondria and unicellular
protozoa.
The universality of the Genetic Code is a result of strong evolutionary
pressure: a change in a single codon would alter nearly every protein
made by an organism.
The universality is the basis for recombinant protein technology:
mammalian mRNA sequences inserted into bacteria will be correctly
expressed (translated). More about this in the last lecture: Expression
Systems for Recombinant Proteins.
Next lecture: tRNA: Structure & Function
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Summary (1.7)
Information Transfer and The Central Dogma
DNA -> DNA
DNA -> RNA
RNA -> Protein
Replication
Transcription
Translation
Substrates: dNTPs (A,T,C,G) Substrates: rNTPs (A,U,C,G)
Substrates : Amino Acids (20)
Chain growth: 5' to 3'
Chain growth: 5' to 3'
Chain growth: N to C
by RNA Polymerase
Step 1: tRNA Synthetases
(different for each AA) use
energy from ATP to couple
amino acids to cognate tRNAs.
by DNA Polymerase
Requires template and primer. Requires only template.
Charging specificity determined
by 3D structural features unique
to each tRNA amino acid pair.
Bases added to 3' OH of
primer according to WatsonCrick pairing with template.
Bases added to 3' OH of growing
chain according to Watson-Crick
pairing with template.
Step 2 by Ribosomes:
Initiated at Promoters.
Initiated at Start Codon (AUG).
In prokaryotes, preceded by
ribosome binding site.
Two classses of RNAs:
Ribosomes provide a platform for
binding of tRNA anticodon to
individual triplet codons in
mRNA according to the Genetic
Code.
1. Messenger (mRNA): single
stranded, rapid turnover.
Amino acids from charged
tRNAs are joined to the carboxyl
end of the growing chain.
Elongation requires GTP
hydrolysis.
Initiated at replication
Origins.
In general double stranded
and stable.
Many mechanisms to assure
fidelity during replication
(proofreading) and
maintenance between
replicative rounds
(recombination and repair).
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Summary (1.7)
2. Stable RNA (eg. tRNA,
ribosomal RNA, snRNAs: folds
into compact structures or
ribonucleoprotein complexes
(RNPs).
Processing:
Processing:
Methylation of bases, ligation Base modification, ligation,
of chains, chain cleavage by
cleavage, splicing & editing,
nucleases. Topological.
polyadenylation, 5' capping.
Processing:
Phosphorylation, acetylation,
chain cleavage by proteases,
disulfide crosslinking, lipidation,
glycosylation etc.
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tRNA: Structure & Function (2.1)
Lecture 2
tRNA: Structure & Function
Major players in protein synthesis:
mRNA, tRNA and the ribosome
mRNA
Messenger RNA, a copy of DNA
blueprint of the gene to be
expressed.
tRNA
Aminoacyl transfer RNA, also
called anticodon or adaptor
molecule. One or more tRNAs
for each amino acid.
Supply
Ribosome
A very large complex of several
rRNAs (ribosomal RNA) and
many protein molecules. Total
molecular weight over 2 million
dalton.
Factory
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Information
tRNA: Structure & Function (2.1)
Protein
Polypeptide chain with sequence
dictated by the mRNA sequence.
Also called the gene product.
Product
Protein Synthesis
Electronmicrograph of a so-called polysome: one mRNA strand
(faint horizontal line) with many individual ribosomes attached
(dark blobs). The newly synthesized polypeptide chains
(proteins) can be seen as irregularly shaped extensions from
the ribosomes:
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tRNA: Structure & Function (2.1)
The bottom panel shows a schematic representation of the
process in the upper panel.
Now let's magnify a ribosome to the size of a "Big Mac" (factor
10,000,000). At this magnification an E. coli bacterium would
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tRNA: Structure & Function (2.1)
be about 10 meters (or 30 feet) in diameter. You would be
about 10,000 miles tall:
Ribosome
20 nm or
200 Å
Big Mac
20 cm
8 inches
tRNA
5 nm or
50 Å
5 cm
2 inches
mRNA (900 bases)
450 nm or
4,500 Å
450 cm
15 feet
Extended (unfolded) protein
(300 amino acids)
90 nm or
900 Å
90 cm
3 feet
Globular (folded) protein
(300 amino acids)
5 nm or
50 Å
5 cm
2 inches
1 nm (nanometer) is 10-9 meters.
1 Å (angstrom) is 10-10 meters or 0.1 nm.
Next: tRNA Structure
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tRNA Structure (2.2)
tRNA Structure
Primary & secondary structure
●
●
●
All tRNAs have similar sequences
of 73 to 93 nucleotides
3' end always terminates with the
sequence CCA, with the 3'
hydroxyl of the ribose of the
terminal A being the point of
covalent attachment of the amino
acid
Contain a number (7-15%) of
unique/modified bases. These are
post-transcriptionally modified
after synthesis by RNA
polymerase.
In particular, adenosine (A)
in first or 5' position of the
anticodon (corresponding to the third or 3' position of the codon) is
always modified to inosine (I) which lacks the amino group on the
❍
●
●
purine ring. Inosine can base-pair with A, U or C and thus
accounts for much of the degeneracy of the Genetic Code
("Wobble Theory").
tRNAs have cloverleaf secondary structure due to four base-paired stems
The cloverleaf contains three non-base-paired loops: D, anticodon, and
TpsiC loop
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tRNA Structure (2.2)
Tertiary structure
●
●
●
●
●
●
●
The tertiary structure of tRNA is best described as a compact "L" shape.
The
anticodon
is a singlestranded
loop at
the
bottom of
the Figure
which
later basepairs with
the triplet
codon
The
amino
acid is
attached
to the
terminal
A on the
upper right.
The active sites (anticodon and amino acid) are maximally separated.
As in proteins, the tertiary structure is dictated by the primary sequence.
The tertiary structure is stabilized by base pairing and base stacking.
Two areas (anticodon stem and acceptor stem) form double helix.
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tRNA Structure (2.2)
Next: tRNA Function
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tRNA Function: Synthetases (2.3)
tRNA Function: Synthetases
●
Each tRNA is charged with the proper amino acid via a covalent ester
bond at their 3' end by a family of enzymes called aminoacyl-tRNA
synthetases. Each enzyme must recognize both the tRNA specific for
an amino acid and the corresponding amino acid. This energyconsuming process is ATP-dependent and results in the cleavage of
two high-energy phosphate bonds (one in going from ATP to AMP +
PP, and one for the cleavage of pyrophosphate into two inorganic
phosphates:
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tRNA Function: Synthetases (2.3)
●
There are 20 different aminoacyl-tRNA synthetases, one for each
amino acid. Despite the fact that they all carry out very similar tasks,
they vary greatly in size (40-100 kDalton).
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tRNA Function: Synthetases (2.3)
●
Since there are 61 amino acid codons, most tRNA synthetases must be
able to recognize more than one type of tRNA (i.e. 6 codons for Arg).
These tRNAs are called cognate tRNAs for that particular synthetase.
This mapping is achieved through so-called recognition domains on
the tRNA. tRNA shown with red backbone and yellow bases. tRNA
synthetase shown as space-filling model in blue:
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tRNA Function: Synthetases (2.3)
●
The recognition domain includes unique of sections of the acceptor
stem and/or the anticodon (black dots):
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tRNA Function: Synthetases (2.3)
Accuracy & Proofreading
●
●
The accuracy of charging tRNA with the proper amino acid is crucial
because once charged, only the tRNA anticodon determines
incorporation, not the attached amino acid.
The error rate of charging is very low: 1 in 10,000. This is achieved
by two means:
❍
❍
the amino acid
specificity pocket in a
specific synthetase will
only bind amino acids
similar in size and
charge.
the synthetase also has
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tRNA Function: Synthetases (2.3)
proofreading capability which, once a wrong aminoacyladenylate complex is formed (1st step), will hydrolyze the
complex before it can be covalently attached to the tRNA (2nd
step).
Next: The Wobble Theory
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The Wobble Theory (2.4)
The Wobble Theory
Assumption:
Each tRNA, defined by its 3-base anticodon, pairs
only with its complementary codon on the mRNA.
Discrepancy:
tRNAAla was found to bind to codons GCA, GCC
and GCU.
Codon (5'->3')
Anticodon (3'->5')
GCA
CGU
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GCC
CGG
GCU
CGA
The Wobble Theory (2.4)
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The Wobble Theory (2.4)
The answer:
The tRNAAla anticodon is actually CGI which pairs
with all three codons.
Experimentally determined codon-anticodon pairing rules:
●
●
●
The first two positions of the mRNA codon observe Watson-Crick base pairing
rules (A-U, C-G)
The third position exhibits wobble.
Wobble occurs because the conformation of the tRNA anticodon loop permits
flexibility at the first base of the anticodon.
5' anticodon base
(tRNA)
A
(not observed)
C
3' codon base
(mRNA)
U
(Watson-Crick)
G
(Watson-Crick)
G
C
or
U
U
A
or
G
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The Wobble Theory (2.4)
I
A
or
C
or
U
Caveat: Since a single tRNA can respond to more than
one codon, one tRNA could respond to codons for two
different amino acids! This would lead to an ambiguous
code. But since the Genetic Code is unambiguous, certain
anticodons are disallowed.
Next: The Dangers of Wobble
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The Dangers of Wobble (2.5)
The Dangers of Wobble
Since a single tRNA can respond to more than one codon, one
tRNA could in theory respond to codons for two different
amino acids:
AGC and AGU both code for Ser. Their anticodon is UCG using wobble
rules. But anticodon UCA would also pair with AGU (Ser). This anticodon (UCA)
would be converted post-transcriptionally to UCI (Hey, that's us!) which would
recognize AGC and AGU as intended, but also AGA which codes for Arg -- not good,
Example:
would result in an ambiguous Genetic Code!
One anticodon for serine is UCG:
Codon (5'->3')
Anticodon (3'->5')
AGC
UCG
AGU
UCG
Amino acid
Ser
Ser
(wobble)
Now, in the following hypothetical example, anticodon UCA (if
it existed) would also base-pair with codon AGU using wobble
rules:
AGU
AGU
UCA => UCI
Ser
Ser
(wobble)
AGC
UCI
AGU
UCI
AGA
UCI
Ser
Ser
Arg
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(wobble)
The Dangers of Wobble (2.5)
But anticodon UCA would be converted post-transcriptionally
to UCI which is able to bind three codons (wobble rules),
including one for Arg! For this reason the UCA/UCI
anticodon does not exist! And the codon AGA for Arg is
actually covered by the anticodon UCU (wobble)!
Next: Supplemental Material
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Supplemental Material (2.6)
Supplemental Material
This page won't be on the final!
Molecular Graphics:
This exercise will let you get a better 3-dimensional picture of the
interaction of a tRNA with its tRNA synthetase. You will be able to rotate,
scale and otherwise manipulate the structure on your screen.
First, you'll need to obtain & install a FREE molecular graphics program called RASMOL on your PC:
Click here
to obtain the MS Windows/NT version
or
Click here
to obtain the Apple Macintosh version
Unpack the distribution, you should then be able to run the program RASWIN (PC) or RASMAC
(MAC). Click here for detailed help. If you get stuck or need help, please contact me (Hudel).
Second, you'll need to get a copy of the atomic coordinates from the Protein Data Bank:
PDB entry 1GTR
Save this rather large file (0.5 MBytes) with the "Save full entry to disk" option. Save the file as
"1GTR.pdb". This file contains the complete atomic coordinates of GLUTAMINYL-tRNA
SYNTHETASE COMPLEXED WITH tRNA AND ATP.
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Supplemental Material (2.6)
Now, run the program RASWIN/RASMAC and go to the "Open..." option under the "File" Menu.
Select the file you just saved (1GTR.pdb). Then select the "Backbone" option under the "Display"
menu. Your display should show something like this. For a description of the program, check the "User
Manual" under the "Help" Menu.
Have fun!
Next: Summary
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Summary (2.7)
Summary
Features of tRNAs
●
●
●
●
●
●
●
tRNAs are the adaptor molecules that translate from nucleic acid
triplet to amino acid.
tRNAs are charged with amino acids by enzymes called aminoacyltRNA synthetases in an energy-dependent 2-step process.
There are 20 specific synthetases, one for each amino acid.
Since most amino acids have more than one possible codon, there are
more than 20 different tRNAs (40 in E. coli).
Not all 61 codons have a specific tRNA due to wobble in the 3rd
codon position.
tRNA synthetases highly specific and have proofreading capabilities,
achieving a better than 1:10,000 error rate.
The accuracy of charging tRNA with the proper amino acid is crucial
because once charged, only the tRNA anticodon determines
incorporation into the growing polypeptide chain, not the attached
amino acid.
Next Lecture: The Ribosome, rRNA and mRNA
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The Ribosome, rRNA and mRNA (3.1)
Lecture 3
The Ribosome, rRNA and mRNA
Major players in protein synthesis:
mRNA, tRNA and the ribosome
mRNA
Messenger RNA, a copy of DNA blueprint of
the gene to be expressed.
tRNA
Aminoacyl transfer RNA, also called anticodon
or adaptor molecule. One or more tRNAs for
each amino acid.
Supply
Ribosome
A very large complex of several rRNAs
(ribosomal RNA) and many protein molecules.
Total molecular weight almost 3 million dalton.
Factory
Protein
Polypeptide chain with sequence dictated by the
mRNA sequence. Also called the gene product.
Product
The Ribosome
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Information
The Ribosome, rRNA and mRNA (3.1)
Ribosomes can be found either free in the cytosol
(cytoplasm) or attached to intracellular membranes.
Free ribosomes
●
●
●
●
Found in the cytosol.
May occur as a single ribosome or in groups known as polyribosomes or polysomes.
Occur in greater number than bound ribosomes in cells that retain most of their manufactured protein.
Responsible for proteins that go into solution in the cytoplasm or form important cytoplasmic structural
or motile elements.
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The Ribosome, rRNA and mRNA (3.1)
Bound ribosomes
●
●
●
Found bound to the exterior of the endoplasmic reticulum (ER) constituting the rough ER.
Occur in greater number than free ribosomes in cells that secrete their manufactured proteins (e.g.,
pancreatic cells, producers of digestive enzymes).
Responsible for proteins that insert into membranes or are packaged into vesicles for storage in the
cytoplasm or export to the cell exterior.
Electronmicrograph of ribosomes (black dots) attached to the rough endoplasmic reticulum.
●
●
●
●
●
●
About 15,000 ribosomes in a single E. coli cell, comprising ~25% of the dry cell mass.
All ribosomes within one cell are identical.
All ribosomes are composed of two subunits (called small and large).
A substantial fraction of ribosomes are dissociated into free subunits in the cell.
Prokaryotic and eukaryotic ribosomes differ in composition.
Mitochondrial ribosomes resemble prokaryotic ribosomes.
Next: The Composition of Ribosomes
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The Ribosome, rRNA and mRNA (3.1)
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The Composition of Ribosomes (3.2)
The Composition of Ribosomes
Prokaryotic ribosomes
●
Sedimentation coefficient: 70S
❍
❍
small subunit: 30S
■ One rRNA molecule (16S)
■ 21 different proteins, designated S1-S21
large subunit: 50S
■ Two rRNA molecules (5S and 23S)
■ 31 different proteins, designated L1-L31 (L12 is present in four copies)
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The Composition of Ribosomes (3.2)
The sedimentation coefficient is measured in Svedberg units (S): the rate of
sedimentation of a component in a centrifuge is related both to the molecular weight and
the 3-D shape of the component.
The rRNA components of the prokaryotic ribosome
Type
Approximate number
of nucleotides
Subunit location
16S
1,542
30S
5S
120
50S
23S
2,904
50S
RNA function and turnover in the cell
Nucleotides
% of
Synthesis
% of
Total
RNA
Stability
Thousands
500-6000
40-50
3
T1/2 = 1-3
min
3 (23S, 16S,
5S)
2904, 1542, 120
50
90
Stable
~50
73-93
3
7
Stable
Different
Kinds
mRNA Messenger
rRNA Ribosome
Type
tRNA
Function
Adapter
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The Composition of Ribosomes (3.2)
Separation of ribosomal proteins by 2D gel electrophoresis
(a) small (30S) subunit; (b) large (50S) subunit
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The Composition of Ribosomes (3.2)
In 1968, Dr. M. Nomura, a professor here at UCI, was the first to show that
the 30S subunit could be reassembled from the individual components.
He found that the order of addition of components was critical.
Eukaryotic ribosomes
●
Sedimentation coefficient: 80S
❍
❍
small subunit: 40S
■ One rRNA molecule (18S)
■ 33 different proteins, designated S1-S33
large subunit: 60S
■ Three rRNA molecules (5S, 5.8S, and 28S)
■ 50 different proteins, designated L1-L50
The rRNA components of the eukaryotic ribosome
Type
Approximate number
of nucleotides
Subunit location
18S
1,900
40S
5S
120
60S
5.8S
156
60S
4,700
60S
28S
Next: Simplified Overview of Translation
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Simplified Overview of Translation (3.3)
Simplified Overview of Translation
1. Formation of the initiation complex
2. Elongation of the polypeptide chain (one repetition of the steps a, b and c
for every amino acid incorporated into the protein being synthesized):
a: binding of the aminoacyl-tRNA
b: peptide bond formation
c: translocation
3. Termination
Rate of synthesis
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Simplified Overview of Translation (3.3)
●
●
Elongation is the rate limiting step in protein synthesis
In E. coli at 37 degrees C:
❍ ribosome passes through 15 codons per second
❍ 300 amino acid polypeptide made in 20 seconds
❍ 15,000 ribosomes per cell can make 750 molecules of a 300 amino acid
protein per second
Next: Structure of the Ribosome
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The Structure of the Ribosome (3.4)
The Structure of the Ribosome
(Prokaryotic ribosomes)
Proposed secondary structure of 16S rRNA
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The Structure of the Ribosome (3.4)
●
●
Many regions are self-complementary and capable of forming double helical segments
Secondary structure is more highly conserved than primary sequence, i.e.
complementary mutations evolve to maintain base paring.
3-Dimensional structure of the 70S ribosome
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The Structure of the Ribosome (3.4)
The large subunit has a tunnel about 10 nm long and 2.5 nm in diameter. This tunnel is
thought to be the channel that newly assembled polypeptide chains pass through on
their way out of the ribosome.
The two subunits interact very tightly and form the active site
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The Structure of the Ribosome (3.4)
Different views: in yellow the 30S (small) subunit;
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in blue the 50S (large) subunit.
The Structure of the Ribosome (3.4)
Landmarks of the 30S subunit: h, head; p, platform; ch, channel
presumed to be the conduit for mRNA; sp, spur.
Landmarks of the 50S subunit: CP, central protuberance; St, L7/L12
stalk; L1, L1 protein; IC, interface canyon; T, tunnel presumed to be the
conduit for the nascent polypeptide chain; T1 and T2, lower tunnel
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The Structure of the Ribosome (3.4)
segments, leading to alternative exit sites E1 and E2, respectively.
Next: The X-ray Structure
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The X-ray Structure of the Ribosome (3.4b)
The X-ray Structure of the Ribosome
Two back-to-back landmark papers in the journal Science (August 2000) provided a whole new level of information
of what ribosomes look like in detail, and specifically, how the crucial peptide bond formation is catalyzed:
The Complete Atomic Structure of the Large Ribosomal Subunit at
2.4 Å Resolution
Nenad Ban, Poul Nissen, Jeffrey Hansen, Peter B. Moore, Thomas A. Steitz
The large ribosomal subunit catalyzes peptide bond formation and binds initiation, termination, and elongation factors. We have
determined the crystal structure of the large ribosomal subunit from Haloarcula marismortui at 2.4 angstrom resolution, and it includes
2833 of the subunit's 3045 nucleotides and 27 of its 31 proteins. The domains of its RNAs all have irregular shapes and fit together in
the ribosome like the pieces of a three-dimensional jigsaw puzzle to form a large, monolithic structure. Proteins are abundant
everywhere on its surface except in the active site where peptide bond formation occurs and where it contacts the small subunit. Most
of the proteins stabilize the structure by interacting with several RNA domains, often using idiosyncratically folded extensions that
reach into the subunit's interior.
Science (2000) 289, 905-920.
The Structural Basis of Ribosome Activity in Peptide Bond Synthesis
Poul Nissen, Jeffrey Hansen, Nenad Ban, Peter B. Moore, Thomas A. Steitz
Using the atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with two substrate analogs,
we establish that the ribosome is a ribozyme and address the catalytic properties of its all-RNA active site. Both substrate analogs are
contacted exclusively by conserved ribosomal RNA (rRNA) residues from domain V of 23S rRNA; there are no protein side-chain
atoms closer than about 18 angstroms to the peptide bond being synthesized. The mechanism of peptide bond synthesis appears to
resemble the reverse of the acylation step in serine proteases, with the base of A2486 (A2451 in Escherichia coli) playing the same
general base role as histidine-57 in chymotrypsin. The unusual pKa (where Ka is the acid dissociation constant) required for A2486 to
perform this function may derive in part from its hydrogen bonding to G2482 (G2447 in E. coli), which also interacts with a buried
phosphate that could stabilize unusual tautomers of these two bases. The polypeptide exit tunnel is largely formed by RNA but has
significant contributions from proteins L4, L22, and L39e, and its exit is encircled by proteins L19, L22, L23, L24, L29, and L31e.
Science (2000) 289, 920-930.
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The X-ray Structure of the Ribosome (3.4b)
The first paper provides a wealth of structural information on how the components of the large (50S) subunit are
arranged:
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The X-ray Structure of the Ribosome (3.4b)
●
●
the rRNA (in gray) is forming the core
the ribosomal proteins (yellow) are mostly on the surface
It also shows the exit tunnel and suggests that not only an extended polpypeptide would fit through it, but also one in
an alpha-helical conformation:
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The X-ray Structure of the Ribosome (3.4b)
The second paper provides a detailed mechanism for peptide bond formation based on the x-ray structure. It
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The X-ray Structure of the Ribosome (3.4b)
highlights the central role of the rRNA, and specifically that of base A2486 (RIBOZYME!):
Next: Supplemental Material
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The X-ray Structure of the Ribosome (3.4b)
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Supplemental Material (3.5)
Supplemental Material
This part won't be on the final!
If you are interested how findings like these are presented in an original
research article, you should take a look at the 1995 paper in the journal
Nature:
"A model of protein synthesis based on cryo-electron microscopy of the
E. coli ribosome"
by Frank, J., Zhu, J., Penczek, P., Li, Y., Srivastava, S., Verschoor, A., Radermacher, M., Grassucci, R.,
Lata, R.K. and Agrawal, R.K.
Nature 376, 441-444 (1995).
And for comparison, check out the more recent back-to-back X-ray papers
in the journal Science:
"The Complete Atomic Structure of the Large Ribosomal Subunit at
2.4 Å Resolution"
by Nenad Ban, Poul Nissen, Jeffrey Hansen, Peter B. Moore, Thomas A. Steitz
Science 289, 905-920 (2000).
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Supplemental Material (3.5)
"The Structural Basis of Ribosome Activity in Peptide Bond Synthesis"
by Poul Nissen, Jeffrey Hansen, Nenad Ban, Peter B. Moore, Thomas A. Steitz.
Science 289, 920-930 (2000).
These issues are available online or as hardcopies at the UCI Science
Library.
Next: Summary
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Summary (3.6)
Summary
●
●
●
●
●
●
●
●
Ribosomes are large and complex molecular precision machines.
Ribosomes occur free in the cytosol or attached to the endoplasmic
reticulum.
They contain two subunits, called small and large.
Both subunits are comprised of large rRNAs and many proteins.
rRNAs form the core and are stabilized by extended base-stacking &
base-pairing.
Most ribosomal proteins are located on the surface.
Peptide-bond formation is catalyzed one base of the 23S rRNA
(ribozyme) in the large subunit.
Ribosomes have tunnels, channels and cavities which accommodate
the various players during translation: mRNA, aminoacyl-tRNA,
nascent protein.
Next Lecture: Initiation of Protein Synthesis
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Initiation of Protein Synthesis (4.1)
Lecture 4
Polypeptide Synthesis Overview
Polypeptide synthesis proceeds sequentially
from N Terminus to C terminus.
Amino acids are not pre-positioned on a template. Proved by the classic experiment of
Dintzis & coworkers in 1961. They synthesized hemoglobin in a test tube using a
reticulocyte (red blood cell) system. They initiated protein synthesis and then added H3labeled leucine. They isolated hemoglobin, partially labeled with incorporated H3-leucine,
and created peptide fragments (labeled a, b, c, d, e, f, g below) by digestion of
hemoglobin with trypsin. Then they analyzed peptide fragments to determine the relative
amount of radioactivity. The following results were obtained:
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Initiation of Protein Synthesis (4.1)
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Initiation of Protein Synthesis (4.1)
Figure 4.1.1: Incorporation of radioactive leucine into hemoglobin after different periods of incubation.
There is a gradient from the C-terminus to the N-terminus of the protein, which implies that synthesis
takes place sequentially from N-terminus to C-terminus. As the time of incubation is increased, the total
3
amount of radioactivity is increased but the gradient remains. Results are expressed as the ratio of an H
14
label (tritium) in leucine to an internal control of C . Data of Dintzis (1961).
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Initiation of Protein Synthesis (4.1)
Ribosomes read mRNA in the 5'->3' direction,
not the 3'->5' direction.
To prove, one must know the Genetic Code. For example, AAA codes for Lys, AAC
codes for Asn and CAA codes for Gln. In a cell-free protein synthesizing system, initiate
protein synthesis by adding artificial mRNA, consisting of polyA with one C (cytosine) at
the 3' end. Analyze the N and C termini of the peptide fragments.
Nucleotide Sequence
Protein Sequence
Direction of Synthesis
5'-AAAAAA(AAA)nAAC-3'
Lys-Lys-(Lys)n-Asn
5' -> 3'
3'-CAAAAA(AAA)nAAA-5'
Gln-Lys-(Lys)n-Lys
3' -> 5'
Next: Polypeptide Synthesis Overview (continued)
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Initiation of Protein Synthesis (4.1)
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Initiation of Protein Synthesis (4.2)
Polypeptide Synthesis Overview (cont.)
Active translation occurs on polyribosomes
Electron micrographs of ribosomes actively engaged in protein synthesis revealed by
"beads on a string" appearance. This implied that each mRNA transcript is read
simultaneously by more than one ribosome. In fact, a second, third, fourth, etc. ribosome
starts to read the mRNA transcript before the first ribosome has completed the synthesis of
one polypeptide chain. Multiple ribosomes on a single mRNA transcript are called
polyribosomes or polysomes. The individual ribosomes are separated by gaps of 50 Å to
150 Å. Thus, there is approximately one ribosome per 80 nucleotides.
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Initiation of Protein Synthesis (4.2)
Chain elongation links the growing polypeptide
to
incoming aminoacyl-tRNA
The newly formed polypeptide chain is attached to a tRNA molecule, not to a protein
or mRNA molecule. To prove, use a cell-free protein synthesizing system with artificial
mRNA, such as polyA. Add H3-Lys. Wash active ribosomes in high salt to dissociate and
stop protein synthesis. Analyze results and find highest concentration of H3-Lys -- always
with tRNA. From many such experiments, there are now known to be three tRNA binding
sites on the ribosomes:
A Site
The ribosomal site which binds the incoming
aminoacyl-tRNA (Acceptor site).
P Site
The site which holds the peptidyl-tRNA, that is the
tRNA which is covalently linked to the growing
polypeptide chain (Peptidyl site).
E Site
A site which transiently binds to the outgoing,
deacylated tRNA (Exit site).
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Initiation of Protein Synthesis (4.2)
Three steps are involved in the synthesis of
one polypeptide chain
1. INITIATION
❍
❍
Assembly of active ribosome and the reading of the first mRNA codon
(START codon).
Occurs once per polypeptide chain.
2. ELONGATION
❍
Involves three distinct steps for each mRNA codon or amino acid in the new
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Initiation of Protein Synthesis (4.2)
polypeptide chain:
1. Transfer of aminoacyl-tRNA from cytoplasm to A-site of ribosome.
2. Covalent linkage of new amino acid to growing polypeptide chain peptidyl transfer.
3. Movement of tRNA from A-site to P-site and simultaneous
movement of mRNA by 3 nucleotides - translocation.
❍
Occurs multiple times per polypeptide.
3. TERMINATION
❍
❍
Reading of final mRNA codon (STOP codon) and dissociation of polypeptide
from ribosome.
Occurs once per polypeptide chain.
Next: Initiation of Protein Synthesis
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Initiation of Protein Synthesis (4.3)
Initiation of Protein Synthesis
INITIATION - Assembly of active ribosome
and the reading of the first mRNA codon.
Occurs once per polypeptide chain.
The signals for initiation:
mRNA Start Codon
Prokaryotes:
Eukaryotes:
AUG (very rarely also GUG & UUG)
AUG
After protein is made, the N-terminal methionine is frequently cleaved off.
Shine-Dalgarno Sequence
To distinguish the start AUG codon from AUG codons which code for
internal methionines or from a fortuitous AUG combination in another
reading frame, the start AUG codon in prokaryotes is preceded by a highly
conserved sequence at the 5´ end of the mRNA transcript which serves as a
ribosome-binding site. The Shine-Dalgarno sequence is a purine-rich tract
of 3-10 nucleotides and precedes the start AUG codon by approximately 10
nucleotides upstream on the 5´ side. The Shine-Dalgarno sequence forms
base pairing with a highly conserved, pyrimidine-rich region of the 16S rRNA
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Initiation of Protein Synthesis (4.3)
which is part of the 30S ribosomal subunit. This base-pairing properly aligns
the start AUG codon in the P site of the 30S ribosomal subunit. In
eukaryotes, protein synthesis usually begins with the first AUG codon from
the 5´ end. Eukaryotic mRNA has a special cap at the 5´ end of the mRNA
which is recognized by a cap-binding protein.
Special Initiator tRNA
Only one special tRNA is used to place the first amino acid, which is always a
formyl-methionine (fMet):
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Initiation of Protein Synthesis (4.3)
The initiator tRNA is called fMet-tRNA. It differs from all other tRNAs by
one less base pair in the acceptor stem region and by three consecutive G:C
base pairs in the anticodon region. These changes alter the 3-dimensional
structure of the fMet-tRNA in subtle ways.
fMet-tRNAfMet and Met-tRNAMet both recognize the AUG codon. A
methionine is attached to both tRNAs by the same methionyl tRNA
synthetase. However, once the methionine is attached to either tRNA, a
special enzyme called formyl transferase, which recognizes only the initiator
tRNAfMet adds a formyl group to the amide nitrogen. Because the amide N is
now blocked from further reaction, this amino acid can only be positioned at
the start of a polypeptide chain.
Soluble Protein Factors
In prokaryotes, there are three initiation factors: IF1, IF2, IF3, named in
order of discovery, not in order of their function on the ribosome. The
initiation factors are proteins that facilitate the assembly of an active
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Initiation of Protein Synthesis (4.3)
ribosomal particle and the placement of the first fMet-tRNAfMet. In
eukaryotes, there are 10 initiation factors, called eIF1, eIF2, etc.
IF3 binds to 30S subunit and promotes the dissociation of the 30S subunit
from the 50S subunit and the subsequent binding of the mRNA. IF1 helps
IF3 by increasing the dissociation rate between the 30S and 50S subunit.
IF2, in complex with GTP, binds to fMet-tRNAfMet and places it in the P-site
of the 30S subunit. Then IF3 is released, causing GTP to hydrolyze to GDP,
which in turn releases IF2-GDP. GTP hydrolysis promotes the release of IF1
and the subsequent association of the 30S subunit with the 50S subunit.
Next: Schematic of Initiation
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Initiation of Protein Synthesis in Prokaryotes (4.4)
Formation of the 70S initiation complex
Schematic of the initiation of protein synthesis in prokaryotes
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Initiation of Protein Synthesis in Prokaryotes (4.4)
1. Binding on IF1 & IF3 to an empty 70S ribosome dissociates it into 50S
and 30S subunits.
2. a. 5' region of mRNA binds to free 30S ribsomal subunit, IF3 is
released.
b. Initiator fMet-tRNAfMet carried by IF2-GTP binds to P site of 30S
ribsomal subunit.
3. Free 50S ribosomal subunit binds, IF1 & IF2-GDP + Pi are released.
Certain details of the process remain uncertain. For example, the exact
order of binding of IF1, IF2, IF3, tRNA, and mRNA is unclear. The above
is one current model (from the text book).
Next: Initiation of translation in eukaryotes
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Initiation of Protein Synthesis in Prokaryotes (4.4)
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Initiation of Protein Synthesis in Eukaryotes (4.5)
Schematic of the initiation of protein synthesis in eukaryotes
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Initiation of Protein Synthesis in Eukaryotes (4.5)
Note that the process is in principle very similar to prokaryotic initiation. Major differences
from prokaryotic initiation are associated with the m7G cap of the mRNA and its interaction
with the cap-binding protein (CBPI) and eIF4F (CBPII) instead of Shine-Dalgarno sequencemediated alignment to properly position the first AUG codon in the P site of the ribosome.
Next: Summary
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Summary (4.6)
Summary
●
●
●
●
●
Polypeptide synthesis proceeds sequentially from N terminus to C
terminus
Ribosomes read mRNA in the 5'->3' direction, not the 3'->5' direction
Active translation typically occurs on polyribosomes
Chain elongation links the growing polypeptide to incoming
aminoacyl-tRNA
Three steps are involved in the synthesis of one polypeptide chain of
N residues:
1. INITIATION (1 x)
2. ELONGATION (N-1 x)
3. TERMINATION (1 x)
●
Initiation in prokaryotes requires:
1. One empty 70S ribosome
2. Three initiation factors: IF1, IF2, IF3
3. mRNA with Shine-Dalgarno sequence and START codon
(AUG)
4. Initiator fMet-tRNAfMet
5. One GTP
●
Initiation in eukaryotes is similar, but more complicated
Next lecture: Elongation & Termination of Protein Synthesis
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Summary (4.6)
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Elongation & Termination of Protein Synthesis (5.1)
Lecture 5
1. INITIATION
●
●
Assembly of active ribosome by placing the first mRNA codon (AUG or START codon) near the
P site and pairing it with initiation tRNA, fMet-tRNAfMet.
Occurs once per polypeptide chain or molecule.
2. ELONGATION
●
Involves three distinct steps for each mRNA codon or amino acid in the new polypeptide chain:
1. Transfer of proper aminoacyl-tRNA from cytoplasm to A-site of ribosome.
2. Covalent linkage of new amino acid to growing polypeptide chain - peptidyl transfer.
3. Movement of tRNA from A-site to P-site and simultaneous movement of mRNA by 3
nucleotides - translocation.
●
Occurs multiple times per polypeptide.
3. TERMINATION
●
●
Reading of final mRNA codon (STOP codon) and dissociation of polypeptide from ribosome.
Occurs once per polypeptide chain.
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Elongation & Termination of Protein Synthesis (5.1)
ELONGATION OF PROTEIN SYNTHESIS
●
●
●
●
●
3 distinct steps to add one amino acid to the growing
polypeptide chain.
Occurs many times per polypeptide, the number of
which depends upon the number of mRNA codons
or amino acids in the protein
The Elongation Cycle is similar in prokaryotes and
eukaryotes.
Fast: 15-20 amino acids added per second
Accurate: 1 mistake every ~10,000 amino acids
Transfer of Aminoacyl-tRNA
Non-initiator, aminoacyl-tRNA is placed in the ribosomal A-site over the mRNA codon in such a
way that base pairing occurs between the anticodon loop of the tRNA and mRNA codon (see
lecture 1).
Aminoacyl-tRNA transfer is facilitated by two soluble protein transfer factors, called elongation
factors, EF-Tu and EF-Ts in prokaryotes:
❍
❍
EF-Tu binds GDP and GTP and is a model G protein.
EF-Ts exchanges GDP for GTP on EF-Tu.
The elongation factors are similar in eukaryotes. Instead of two proteins, there is a stable trimer,
eEF1-alpha-beta-gamma, which carries out the same function as EF-Tu and EF-Ts. eEF1-alpha is
the eukaryotic equivalent of EF-Tu, and eEF1-beta-gamma the eukaryotic equivalent of EF-Ts.
EF-Tu-GDP is the inactive form. EF-Ts activates EF-Tu by catalyzing the exchange of GDP for
GTP. EF-Tu-GTP is the active form which binds to non-initiator tRNAs to which the aminoacyl
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Elongation & Termination of Protein Synthesis (5.1)
group has been attached.
EF-Tu-GTP-aminoacyl-tRNA is then carried to the ribosome. The complex binds to the
ribosome, with the aminoacyl-tRNA in the A-site. Ribosome binding stimulates GTP hydrolysis
and EF-Tu-GDP dissociates from the ribosome, free to recycle through the step multiple times.
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Elongation & Termination of Protein Synthesis (5.1)
EF-Tu is partially responsible for the high degree of accuracy of protein synthesis via a
mechanism called kinetic proofreading. The error rate of protein synthesis is only 1 wrong amino
acid for every 10,000 amino acids added to polypeptides.
When a charged tRNA is positioned into the A-site, the anticodon must base pair with the mRNA
codon. If there is an incorrect match, the incorrect aa-tRNA dissociates from the ribosome before
GTP hydrolysis occurs. If there is a correct match, GTP hydrolysis occurs and EF-Tu-GDP leaves
the ribosome before the cognate aminoacyl-tRNA can dissociate and EF-Tu-GDP dissociates
instead, leaving the correct tRNA on the ribosome.
EF-Tu is so important to cellular function that it is one of the most abundant cytoplasmic proteins
(>5%). There is one copy of the EF-Tu protein for each tRNA molecule in the cell.
Next: Peptidyl Transfer
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Elongation & Termination of Protein Synthesis (5.1)
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Elongation & Termination of Protein Synthesis (5.2)
ELONGATION OF PROTEIN SYNTHESIS (cont.)
Peptidyl Transfer or Transpeptidation
Next, the growing polypeptide chain on the P-site tRNA (peptidyl tRNA) is covalently linked to the amino acid
attached to the A-site tRNA, that is a peptide bond is formed. The P-site tRNA is now unacylated and the A-site tRNA
is covalently linked to the growing polypeptide chain. This step is somewhat of a mystery but is believed to be
catalyzed by a region of the 50S subunit, called the peptidyltransferase complex. The complex utilizes both proteins
and 23S rRNA. There is growing evidence that 23S rRNA actively catalyzes the peptidyl transfer step but the mode of
action is yet unknown.
Translocation
The A-site tRNA with the growing polypeptide chain is then moved within the ribosome to the P-site while the
deacylated P-site tRNA is moved to the E-site. Simultaneously, the mRNA is shifted by exactly 3 ribonucleotides or 1
codon.
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Elongation & Termination of Protein Synthesis (5.2)
The process requires a soluble protein factor, called elongation factor EF-G. EF-G is similar, but much larger than EFTu. EF-G is also active as the GTP complex.
Although many older texts report that the binding of EF-G-GTP to the ribosome provides the energy for
translocation, it is now known to be wrong. The energy derived from GTP hydrolysis drives translocation.
After GTP hydrolysis, EF-G-GDP is released from the ribosome, the tRNA carrying the polypeptide chain is in the P
site, and the next mRNA codon is in the A site. The ribosome is now ready to start the elongation cycle over again to
add a new amino acid, until a termination signal is reached. It appears that EF-G catalyzes its own exchange of GDP
and GTP. No soluble guanine nucleotide exchange factor has yet been found. The equivalent of EF-G in eukaryotes
is eEF2.
Next: Schematic of Elongation
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Elongation & Termination of Protein Synthesis (5.3)
Chain elongation in prokaryotic translation
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Elongation & Termination of Protein Synthesis (5.3)
The elongation process is depicted as a cycle. Following translocation (step 3) and
empty tRNA release (step 4), the ribosome is ready to accept the next aminoacyl
tRNA (aa-tRNA) and repeat the cycle. This cycle will continue until a termination
codon is reached.
Next: Atomic Structures of EF-Tu Complexes
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Elongation & Termination of Protein Synthesis (5.4)
ATOMIC STRUCTURES OF EF-Tu COMPLEXES
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Elongation & Termination of Protein Synthesis (5.4)
Next: Termination of Protein Synthesis
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Elongation & Termination of Protein Synthesis (5.5)
TERMINATION OF PROTEIN SYNTHESIS
1. The mRNA Signal
STOP Codons:
UAA, UAG, or UGA
There are no tRNAs that recognize the STOP codons UAA, UAG, or UGA.
2. Soluble Protein Release Factors
RF1 responds to UAA or UAG
RF2 responds to UAA or UGA
RF3, a GTPase (like EF-Tu and binds in a similar A-site location)
RF1/RF2 interact with RF3-GTP, have a similar shape as EF-Tu-GTP-aa-tRNA or
EF-G, and bind in a similar ribosomal site (A-site). In a manner similar to EF-G,
GTP hydrolysis drives the movement of the terminal mRNA codon into the P-site,
moving the last tRNA into the E-site and off. At the same time, the polypeptide
chain is released after hydrolysis of the tRNA-peptide bond.
In eukaryotes, only a single release factor, eRF, is necessary. It recognizes all three
STOP codons and interacts with GTP.
A mutation resulting in a premature STOP codon is called a nonsense mutation.
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Elongation & Termination of Protein Synthesis (5.5)
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Elongation & Termination of Protein Synthesis (5.5)
The termination pathway in E. coli ribosomes: RF-1 recognizes the termination codons
UAA and UAG, whereas RF-2 recognizes UAA and UGA. Eukaryotic termination
follows an analogous pathway but requires only a single release factor, eRF, that
recognizes all three termination codons.
Next: Summary
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Summary (5.6)
Summary
●
●
●
●
●
●
●
●
●
●
●
●
Elongation consists of three distinct steps to add one amino acid
Requires three elongation factors: EF-Tu/EF-Ts and EF-G
Requires two GTPs per cycle
Occurs many times per polypeptide
The elongation cycle is similar in prokaryotes and eukaryotes.
Fast: 15-20 amino acids added per second
Accurate: 1 mistake every ~10,000 amino acids
Termination results in the release of the polypeptide chain
Requires one of the three STOP codons: UAA, UAG, or UGA.
Requires RF1 or RF2, and RF3 in prokaryotes (eRF in eukaryotes)
Requires one GTP
Each step of protein synthesis (initiation, elongation and termination)
requires GTP
Next lecture: Regulation of Protein Synthesis at the
Translational Level
And now: Movie time!
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Summary (5.6)
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Regulation of Protein Synthesis (6.1)
Lecture 6
Regulation of Protein Synthesis at the Translational Level
Comparison of EF-Tu-GDP and EF-Tu-GTP conformations
EF-Tu-GDP
EF-Tu-GTP
Next: Comparison of GDP and GTP binding region in EF-Tu
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Regulation of Protein Synthesis (6.1a)
Comparison of GDP and GTP binding region of EF-Tu
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Regulation of Protein Synthesis (6.1a)
Next: Molecular Mimicry
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Regulation of Protein Synthesis (6.1b)
Molecular mimicry between
EF-G and EF-Tu-GTP-aminoacyl-tRNA
EF-G
EF-Tu-GTP-Aminoacyl-tRNA
Next: Rates and Energetics of Translation
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Regulation of Protein Synthesis (6.2)
Rates and energetics of translation
At 37° C, the rate of translation in E. coli is about 15 amino acids
per second.
The translational rate is equivalent to the transcriptional rate which
is ~45 nucleotides per second.
Energy cost for synthesis of a protein with N amino acids:
2N
1
ATPs to charge tRNA (ATP -> AMP + PP -> AMP + 2Pi)
GTP for initiation (IF2)
N-1
GTPs to position tRNA for N-1 peptide bonds (EF-Tu)
N-1
GTPs for N-1 translocation steps (EF-G)
1
GTP for termination (RF-3)
====
4N
Total of 4 high-energy phosphate bonds cleaved per amino acid
Each ATP or GTP cleavage generates ~40 kJ/mol
Each peptide bond costs ~160 kJ/mol in the cell, yet an uncatalyzed chemical
reaction to form a peptide bond costs only ~20 kJ/mol.
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Regulation of Protein Synthesis (6.2)
Why is it so costly to make a peptide bond on a ribosome?
The excess energy is used for generating an accurate, defined
polypeptide sequence, not a random one or a combination of
multiple possibilities.
Two sources of errors during translation:
●
●
Attachment of an incorrect amino acid to a tRNA
Mispairing of the tRNA anticodon with the mRNA codon
Two proofreading mechanisms exist to prevent these errors:
●
●
Proofreading before aminoacyl adenylate intermediate is attached to tRNA.
Kinetic proofreading before peptide bond formation: A delay is introduced
between the binding of an aminoacyl-tRNA to the codon and the formation
of the peptide bond to allow errors to be corrected:
EF-Tu-GTP binds an aminoacyl-tRNA and bring it into the A-site.
EF-Tu allows the anticodon to interact with the codon but prevents
peptide bond formation.
An incorrect tRNA will bind weakly to the codon and will dissociate
from the codon before an incorrect amino acid is incorporated into the
polypeptide.
Correct codon-anticodon matching triggers hydrolysis of GTP by the
EF-Tu, after which EF-Tu-GDP dissociates.
Peptide bond formation proceeds.
❍
❍
❍
❍
❍
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Regulation of Protein Synthesis (6.2)
Each major step in protein synthesis, except peptide-bond
formation itself, involves hydrolysis of GTP to GDP.
Regulation of protein synthesis
PROKARYOTES
Short-lived mRNA (few minutes), so little need for complicated
translational regulation. In prokaryotes, most of the regulation is at the
transcriptional level.
Rates vary only by a factor of 100. Variance is due to differences in ShineDalgarno sequences and how strongly a particular sequence base-pairs with
the 16S rRNA of the 30S ribosomal subunit.
EUKARYOTES
Long-lived mRNA (hours to days) and thus a greater need to
regulate the rate of protein synthesis.
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Regulation of Protein Synthesis (6.2)
Several known mechanisms:
●
●
mRNA masking: mRNA is bound to a variety of proteins that prevent
association with ribosomes. When appropriate signal is received, the
proteins dissociate from mRNA, leaving the transcript free to associate with
the ribosome. The signal is usually in the form of phosphorylation/
dephosphorylation. mRNA masking is a major form of regulation in early
embryonic development.
antisense RNA: short segment of RNA, complementary to mRNA, that
forms double stranded RNA which cannot be translated by ribosome. Two
known examples:
❍
❍
●
blockage of protein synthesis of fruit-ripening enzyme in tomatoes
the c-myb gene product which promotes smooth muscle development
and blockage in injured arteries.
Heme Control of Globin Synthesis: Red blood cells are programmed to
synthesize large amounts of globin. The globin chains, subsequent to
translation, are assembled with heme into hemoglobin. If there is an
insufficient supply of heme to insert into the newly synthesized globin
chains, then translation is turned off. The lack of heme triggers the
accumulation of a heme-controlled inhibitor (HCI) protein. This protein is
a kinase which phosphorylates eIF2-GTP. The phosphorylation blocks the
dissociation of eIF2 and eIF2-beta that normally occurs in the initiation
cycle. Thus, the cell becomes rapidly depleted of unphosphorylated eIF2
which is normally recycled for initiation of additonal mRNA. Either the
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Regulation of Protein Synthesis (6.2)
addition of heme, which represses the production of HCI, or the addition of
lots of unphosphorylated eIF2, which bypasses the HCI effect, can restart
initiation again.
●
Interferon: Interferons are glycoproteins that are secreted by virusinfected cells. Interferons prevent additional infection by other types of
viruses by inhibiting protein synthesis in infected cells.
Two mechanisms of action:
❍
❍
induces production of protein kinase, DAI (double-stranded RNAactivated inhibitor) which in the presence of dsRNA, phosphorylates
eIF2-alpha and stabilizes the eIF2-alpha-eIF2-beta complex in a
manner similar to the heme-controlled inhibitor (HCI).
induces a cascade effect which ultimately activates an endonuclease,
RNase L, that rapidly degrades mRNA.
Inhibition of protein synthesis by antibiotics
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Regulation of Protein Synthesis (6.2)
Antibiotics are bacterially or fungally produced substances that inhibit the
growth of other organisms. Antibiotics target a wide spectrum of vital
processes: they block DNA replication, transcription and bacterial cell wall
synthesis. A large number of antibiotics, including medically useful substances,
block protein translation.
Blocking protein translation is very effective for two reasons:
●
●
Protein translation plays a central role in overall metabolism
The structural differences between prokaryotic and eukaroytic ribosomes
and associated factors (IFs/EFs/RFs) allow specific targeting.
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Regulation of Protein Synthesis (6.2)
Prokaryotic Inhibitors
●
●
●
●
●
●
Chloramphenicol - inhibits peptidyl transferase on 50S subunit.
Erythromycin - inhibits translocation by 50S subunit.
Fusidic acid - inhibits translocation by preventing the dissociation of EF-GGDP from ribosome.
Puromycin - an aminoacyl-tRNA analog that causes premature chain
termination.
Streptomycin - causes mRNA misreading and inhibits chain initiation.
Tetracycline - inhibits binding of aminoacyl-tRNA to ribosomal A-site.
Eukaryotic Inhibitors
●
●
●
Puromycin & Tetracycline (see prokaryotes above).
Cycloheximide - inhibits peptidyl transferase on 60S subunit.
Diphtheria Toxin - inactivates eEF-2 by ADP ribosylation.
Summary
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Summary (6.3)
Summary
●
●
●
●
●
●
●
●
GTP-binding often causes major conformational changes in
proteins
Each major step in protein synthesis, except peptide-bond
formation itself, involves hydrolysis of GTP to GDP.
Four high-energy phosphate-bonds are required for each amino
acid added
The rate of protein synthesis is well matched to the rate of
transcription
EF-G is shaped like EF-Tu-GTP-aminoacyl-tRNA (molecular
mimicry)
mRNA in prokaryotes is short-lived (minutes)
mRNA in eukaryotes is long-lived (hours/days), requiring
additional control
A large number of antibiotics inhibit protein synthesis, many
specifically in prokaryotes
Next lecture: Post-Translational Processing
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Post-Translational Processing (7.1)
Lecture 7
Post-Translational Processing
Protein synthesis consists of several steps: from
the translation of the information from mRNA
to the folded and fully processed, active protein
in its proper compartment of action.
The mRNA sequence predicts a specific length
polypeptide chain made up of the primary 20
amino acids.
Fully processed protein products are almost
always shorter than their mRNA would predict,
and globally contain about 200 different amino
acids.
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Post-Translational Processing (7.1)
(determined by sequencing, biochemistry, X-ray crystallography)
During translation, about 30-40 polypeptide residues are
relatively protected by the ribosome (tunnel T and exit sites E1
and E2 in the large subunit). Once the polypeptide chain
emerges from the ribosome it starts to fold and can be subject
to post-translational modifications.
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Post-Translational Processing (7.1)
So after translation several additional steps must be
considered as part of the complete protein biosynthetic
process:
1. Covalent modification of
a:
b:
c:
d:
peptide bonds
the N-terminus
the C-terminus
amino acid residues (side chains).
2. Noncovalent modifications: folding, addition of co-factors.
3. Translocation: compartment selection and transport (Trafficking/Targeting).
4. Involvement of molecular chaperones in 1, 2, and 3.
Why post-translational processing?
●
adds functionality
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Post-Translational Processing (7.1)
●
●
●
●
effects targeting
regulates activity
increases mechanical strength
changes recognition
Next: Covalent Modifications
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Covalent Modifications (7.2)
Covalent Modifications
Modifications involving the peptide bond (peptide bond cleavage or
limited proteolysis):
●
usually carried out by enzymes called peptidases or proteases:
❍
❍
❍
❍
activation of proenzymes (digestive enzymes, blood clotting cascade, complement
activation etc.) and prohormones (insulin)
production of active neuropeptides and peptide hormones from high molecular weight
precursors
macromolecular assembly in virus particles (e.g. HIV protease)
removal of signal sequences
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Covalent Modifications (7.2)
These reactions are often exquisitely specific for only
one or a few peptide bonds.
Modifications involving the amino terminus:
●
●
●
●
trimming of formyl group from formyl-Met
proteolytic removal of N-terminal Met by aminopeptidases
acetylation
lipidation (myristoylation)
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Covalent Modifications (7.2)
Modifications involving the carboxy terminus:
●
●
amidation of C-terminal glycine
attachment of membrane anchors
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Covalent Modifications (7.2)
Next: Side Chain Modifications
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Side Chain Modifications (7.3)
Side Chain Modifications
Modifications involving amino acid side chains:
●
disulfide cross-linking
●
lysinonorleucine cross-linking (collagen)
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Side Chain Modifications (7.3)
●
Phosphorylation of hydroxyls by kinases (serine, threonine, tyrosine):
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Side Chain Modifications (7.3)
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Side Chain Modifications (7.3)
Glycogen phosphorylase is the ultimate enzyme in a cascade which catalyzes the degradation of
glycogen to glucose-1-phosphate. Phosphorylation of phosphorylase occurs on serine-14 and
converts the inactive phosphorylase b to the active phosphorylase a.
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Side Chain Modifications (7.3)
Phosphorylation is reversible and is used in many
pathways to control activity. Enzymes that add a
phosphate to a hydroxyl side chain are commonly called
kinases. Enzymes that remove a phosphate from a
phosphorylated side chain are called phosphatases.
Glycosylation
●
There are two basic types of glycosylation which occur on:
asparagines (N-linked, see (a) below) and
serines and threonines (O-linked, see (b) below)
❍
❍
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Side Chain Modifications (7.3)
(b) N-Acetylgalactosamine
●
●
Covalently attached to the polypeptide as oligosaccharide chains containing 4 to 15 sugars
Sugars frequently comprise 50% or more of the total molecular weight of a glycoprotein
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Side Chain Modifications (7.3)
●
●
●
●
Most glycosylated proteins are either secreted or remain membrane-bound
Glycosylation is the most abundant form of post-translational modification
Glycosylation confers resistance to protease digestion by steric protection
Important in cell-cell recognition
N-linked glycosylation on asparagine (Asn) side chains:
●
●
●
●
●
●
an alkali-stable bond between the amide nitrogen of asparagine and the C-1 of an amino
sugar residue
occurs co-translationally in the endoplasmic reticulum (ER) during synthesis
lipid-linked oligosaccharide complex is transferred to polypeptide by oligosaccharyl
transferase
target sequence or consensus site on protein is Asn-X-Ser/Thr
further processing in Golgi apparatus
Examples:
❍ Heavy chain of immunoglobulin G (IgG)
❍ Hen ovalbumin
❍ Ribonuclease B
O-linked glycosylation on serine (Ser) or threonine (Thr) side chains
●
●
●
●
an alkali-labile bond between the hydroxyl group of serine or threonine and an amino sugar
carried out by a class of membrane-bound enzymes called glycosyl transferases which
reside in the endoplasmic reticulum (ER) or the Golgi apparatus
nucleotide-linked monosaccharides added to protein side chain one at a time
Example: Blood group antigens on erythrocyte surface:
❍ The A antigen and B antigen are pentasaccharides which differ in composition of the
5th sugar residue
th residue and does not
❍ The O substance is a tetrasaccharide which is missing the 5
elecit an antibody response (non-antigenic).
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Side Chain Modifications (7.3)
,
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Side Chain Modifications (7.3)
Blood
Type
Glycolsyl transferase Antibodies Against
(Antigen)
Can Safely
Receive Blood
from
Can Safely
Donate Blood to
O
neither
A&B
O
O, A, B & AB
A
UDP-GalNAc
B
O&A
A & AB
B
UDP-Gal
A
O&B
B & AB
AB
both
neither
O, A, B, & AB
AB
Examples of monosaccharides used in glycosylation
●
Prosthetic group attachment (heme, retinal etc.)
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Side Chain Modifications (7.3)
Heme group, attached to histidine side chain via Fe.
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Side Chain Modifications (7.3)
Retinal attached to lysine side chain via covalent Schiff base linkage.
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Side Chain Modifications (7.3)
Processing of pre-pro-insulin to active insulin
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Side Chain Modifications (7.3)
●
●
●
●
Pre-pro-insulin is synthesized as a random coil on membrane-associated
ribosomes
After membrane-transport the leader sequence (yellow) is cleaved off by a
protease and the resulting pro-insulin folds into a stable conformation.
Disulfide bonds form between cysteine side chains.
The connecting sequence (red) is cleaved off to form the mature and active
insulin molecule.
Next: Noncovalent Modifications
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Noncovalent Modifications (7.4)
Noncovalent Modifications
Addition of metal ions and co-factors:
Nearly 50% of all proteins contain metal ions
Metal ions play regulatory as well as structural roles
●
●
●
●
Calcium (Ca++): very important intra-cellular messenger, i.e. calmodulin
Magnesium (Mg++): ATP enzymes
Copper (Cu++), Nickel (Ni+), Iron (Fe++)
Zinc (Zn++): Zinc finger domains are used for DNA recognition:
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Noncovalent Modifications (7.4)
A zinc finger domain: Zn++ is bound by two cysteine and two histidine residues. Zinc finger
domains interact in the major groove with three consecutive bases from one strand of duplex Bform DNA.
Modifications involving tertiary structure (protein fold)
Enzymes called molecular chaperones are responsible for detecting mis-folded proteins.
Chaperones only bind mis-folded proteins that exhibit large hydrophobic patches on their
surfaces.
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Noncovalent Modifications (7.4)
Subunit multimerization
Many enzymes are only functional as multimeric units, either as homo- or hetero-oligomers.
Example: ribosomes!
Next: Chaperone-Assisted Protein Folding
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Chaperone-Assisted Protein Folding (7.5)
Chaperone-Assisted Protein Folding
Quote: "The traditional role of the human chaperone described in biochemical terms is to
prevent improper interactions between potentially complementary surfaces, and to disrupt
any improper liaisons which occur."
Chaperones:
●
●
●
●
●
●
●
●
●
Mediate folding and assembly.
Do not convey steric information.
Do not form part of the final structure.
Suppress non-productive interactions by binding to transiently exposed portions of
the polypeptide chain.
First identified as heat shock proteins (Hsp).
Hsp expression is elevated when cells are grown at higher-than-normal temperatures.
Stabilize proteins during synthesis.
Assist in protein folding by binding and releasing unfolded/mis-folded proteins.
Use an ATP-dependent mechanism.
Major types of chaperones:
●
Hsp70 (cytoplasm, ER, chloroplasts, mitochondria):
❍ thought to bind and stabilize the nascent polypeptide chain as it is being
extruded from the ribosome.
❍ also involved in "pulling" newly synthesized polypeptide into ER lumen.
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Chaperone-Assisted Protein Folding (7.5)
●
Hsp60 (mitochondria, chloroplasts):
❍ forms large 28-subunit complexes called GroEL
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Chaperone-Assisted Protein Folding (7.5)
Next: Selenocysteine
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Selenocysteine (7.6)
Selenocysteine: the 21st Amino Acid?
Although the element selenium was discovered in 1817 by Berzelius
it was only shown over 100 years later to be an essential micronutrient
in all three lines of decent. Subsequent analysis of several enzymes
that catalyze oxidation-reduction reactions showed that selenium
occurs in the form of the unusual amino acid selenocysteine. How
this amino acid is incorporated into the protein was unclear in these
days.
Today it is well established that the incorporation of selenocysteine is
co-translational. Interestingly, the base triplet encoding this amino
acid is UGA, a codon that normally functions as a STOP signal in
translation. Since this codon has still retained its "normal" function in
all organisms known to synthesize selenocysteine-containing proteins,
the incorporation of this amino acid requires a specific pathway. This
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Selenocysteine (7.6)
pathway has been elucidated for the bacterium E. coli by August Böck
and coworkers.
General pathway
There are one cis-acting element and four trans-acting factors
involved in this incorporation. The cis-acting element is a specific
stem-loop structure of the mRNA directly 3' of the UGA codon. The
four trans-acting factors are:
●
●
●
●
a specific tRNASec which is charged with serine by serinyl-tRNA
synthetase
the enzyme selenophosphate synthetase which generates inorganic
selenophosphate
the enzyme selenocysteine synthetase which uses selenophosphate to
convert seryl-tRNASec to selenocysteyl-tRNASec
a specific translation factor (SelB) that substitutes for EF-Tu and
recognizes the cis-acting element
In the first step the specific tRNASec is charged by the normal seryltRNA synthetase with serine, and that serine is subsequently
converted to selenocysteine by the enzyme selenocysteine synthetase.
The low molecular weight selenium donor - selenophosphate - is
provided by the action of the selenophosphate synthetase. Finally, the
selenocysteyl-tRNASec is recognized by a specific translation factor
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Selenocysteine (7.6)
SelB that delivers it to a UGA codon at the ribosomal A-site only in
the presence of the stem-loop structure downstream of the codon on
the mRNA.
tRNASec
tRNASec differs in several aspects from the consensus for canonical
tRNAs. In particular it includes an acceptor stem elongated by extra
one base pair, an elongated D-arm with only four nucleotides in the
loop, and a UCA anticodon. These differences ensure that tRNASec is
not recognized by the normal translation factor EF-Tu thus preventing
mis-incorporation.
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Selenocysteine (7.6)
The two structures shown here are (a) tRNASec and (b) tRNASer of E. coli. Residues which are
normally conserved in tRNA are shown circled. Identity nucleotides in tRNASer and their
counterparts in tRNASec are shown boxed. tRNASec is the longest tRNA species known
consisting of 95 nucleotides.
Selenophosphate synthetase
This enzyme catalyses the synthesis of the low molecular weight
donor selenophosphate using ATP as the phosphate donor.
Interestingly, the gamma-phosphate is transferred to selenium
whereas the beta-phosphate is released, leaving AMP.
Selenocysteine synthetase
This enzyme possesses a prosthetic group (pyridoxal phosphate). The
active enzyme is composed of ten identical subunits arranged as a
stack of two five-membered rings. It converts the serine attached to
tRNASec to selenocysteine.
Translation factor SelB
This translation factor substitutes for elongation factor EF-Tu in the
specific incorporation of selenocysteine. This protein is considerable
longer than its regular counterpart. The N-terminal half of the protein
resembles EF-Tu in structure and function - GTP and tRNA binding whereas the C-terminal half is responsible for the recognition of the
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Selenocysteine (7.6)
specific stem-loop structure adjacent to the UGA codon. A complex
between SelB, GTP, selenocysteyl-tRNASec and the stem-loop
structure of the mRNA has been detected and shown to be
functionally necessary.
Next: Summary
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Summary (7.7)
Summary
●
●
●
Post-translational processing controls folding, targeting, activation
and stability of proteins
Co- and post-translational modifications increase diversity and
functionality of proteins
Common forms of co- or post-translational modifications are:
proteolysis
phosphorylation
glycosylation
metal binding
❍
❍
❍
❍
●
●
Chaperones mediate folding and assembly of newly synthesized
proteins
Selenocysteine is sometimes called the 21st amino acid
uses one of the three STOP codons
has its own tRNASec
requires other factors, including a cis-acting element on the
mRNA
❍
❍
❍
Next Lecture: Protein Trafficking/Targeting
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Protein Trafficking/Targeting (8.1)
Lecture 8
Protein Trafficking/Targeting
Protein targeting is necessary for proteins that
are destined to work outside the cytoplasm.
Protein targeting is more complex in eukaryotes
because of the presence of many intracellular
compartments.
Prokaryotic protein targeting (secretion)
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Protein Trafficking/Targeting (8.1)
The chaperone protein SecB binds to the nascent polypeptide chain to prevent
premature folding which would make transport across the plasma membrane
impossible. SecE and SecY are transmembrane components which form a pore in
the membrane through which the still unfolded polypeptide is threaded. The
translocation process is energy-dependent (ATP) and is driven by SecA. Once the
protein has passed through the pore, the signal sequence is cleaved off by an
extracellular, membrane-bound protease.
N-terminal signal sequences of representative secreted prokaryotic
proteins.
Protein
-20
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-10
-1 +1
Protein Trafficking/Targeting (8.1)
Leucinebinding
protein
MKANAKTIIAGMIALAISHTAMA
EE...
Pre-alkaline
phosphatase
MKQSTIALALLPLLFTPVTKA
RT...
Prelipoprotein
MKATKLVLGAVILGSTLLAG
CS...
Hydrophobic residues in red.
Next: Eukaryotic Protein Targeting
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Eukaryotic Protein Targeting (8.2)
Eukaryotic Protein Targeting
Targeting in eukaryotes is necessarily more complex due to the
multitude of internal compartments:
●
●
●
●
●
●
●
●
nucleus
mitochondria
peroxisomes
chloroplasts
endoplasmic reticulum (ER)
Golgi
lysosomes
secretory granules
The signals involved are also called sorting signals.
They are regions on the targeted protein with certain
amino acid sequences.
These signals interact with specific receptors, either
on the target organelle or a carrier protein.
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Eukaryotic Protein Targeting (8.2)
There are two basic forms of targeting pathways:
●
●
post-translational targeting:
❍ nucleus
❍ mitochondria
❍ chloroplasts
❍ peroxisomes
co-translational targeting (secretory pathway):
❍ ER
❍ Golgi
❍ lysosomes
❍ plasma membrane
❍ secreted proteins
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Eukaryotic Protein Targeting (8.2)
In the absence of targeting signals, a protein will remain in the
cytoplasm:
●
●
●
●
translational machinery
metabolic enzymes
cytoskeletal proteins
many signal transduction proteins
Nuclear targeting:
●
Unusual since 2-way traffic:
❍ in: proteins, DNA
■ DNA & RNA polymerases
■ transcriptions factors
■ histones etc.
❍ out: mRNA, tRNA, rRNA
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Eukaryotic Protein Targeting (8.2)
●
Proteins are not transported through the nuclear membrane but rather through a
complex pore called the nuclear pore:
❍ comprised of about 100 different proteins
❍ proteins smaller than 20 kDa move by diffusion
❍ proteins larger than 20 kDa move by selective transport (nuclear localization
signal)
■ cluster of 4-8 positively charged amino acids (example: PKKKRLV)
■ signal sequence binds to receptor on the pore called importin
Mitochondrial targeting:
●
●
not well understood
usually by post-translational targeting
Lysosomal targeting:
●
●
●
●
Lysosomes are organelles that store enzymes which rapidly degrade other proteins
and nucleic acids.
A famous target sequence is "KDEL"
Initial targeting via secretory pathway
Final targeting occurs in the Golgi
Next: The Secretory Pathway
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The Golgi (8.2b)
The secretory pathway
ER targeting (secretory pathway)
●
co-translational insertion of protein into or through ER membrane via attached ribosomes (rough ER):
❍ signal sequence of 16-30 amino acids at N-terminus (hydrophobic)
❍ emerging signal sequence of nascent protein on free ribosome binds to signal recognition particle (SRP) -- translation is arrested.
■ SRPs consist of 6 proteins and one RNA molecule (7S RNA).
■ The SRP-signal sequence-mRNA-ribosome complex docks with receptor on ER membrane.
❍ signal sequence crosses ER membrane.
❍ translation continues with polypeptide chain being pulled into the ER lumen.
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The Golgi (8.2b)
While in the ER, many proteins undergo the first stages of glycosylation. Most proteins then migrate inside vesicles from the ER and enter the cis face of the Golgi where further
processing and final sorting occurs:
The Golgi Complex
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The Golgi (8.2b)
The Golgi is responsible for further processing and final sorting of proteins. One example is the formation of primary and secondary lysosomes:
●
Primary lysosomes bud from the trans face of the Golgi and subsequently
❍ undergo exocytosis (A)
❍ fuse with vesicles to digest their contents (B & C)
❍ rupture, causing autolysis (D)
Overview of Trafficking
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The Golgi (8.2b)
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The Golgi (8.2b)
Next: Targeted Protein Degradation
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Targeted Protein Degradation (8.3)
Targeted Protein Degradation
Why are proteins degraded?
In order to keep a cell working it needs to remove:
●
●
●
●
incorrectly synthesized proteins (with errors in amino acid sequence)
damaged proteins (i.e. oxidative damage)
cell-cycle specific proteins
other signaling proteins which are no longer necessary
One mechanism of protein degradation is via lysosomes.
Lysosomes are acidic vesicles that contain about 50 different
enzymes involved in degradation:
●
●
●
●
●
●
●
●
proteases (cathepsins): cleave peptide bonds
phosphatases: remove covalently bound phosphates
nucleases: cleave DNA/RNA
lipases: cleave lipid molecules
carbohydrate-cleaving enzymes: remove covalently bound sugars from
glycoproteins
Lysosomes often secrete their contents into the extracellular medium via exocytosis.
Lysosomes can also target damaged organelles in a process called autophagy.
Sometimes, lysosomes are triggered to rupture inside a cell, resulting in autolysis,
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Targeted Protein Degradation (8.3)
also called apoptosis or programmed cell death.
Another major mechanism is via ubiquitin labeling of surplus
proteins:
●
●
●
●
Ubiquitin (a small 76-residue protein) is attached to the protein:
❍ First, an activating enzyme attaches itself to the carboxy terminus of free
ubiquitin in an ATP-dependent process.
❍ Then, the activated ubiquitin is transferred onto a second enzyme which at the
same time recognizes damaged proteins.
❍ The activated ubiquitin is then covalently linked to lysine residues on the
surface of the damaged protein.
These ubiquitin-tagged proteins are now recognized by specific proteases in the
cytosol which in turn cleave and degrade the tagged protein.
These proteases are combined in a very large protein complex called the proteasome.
The proteasome (20S) is comprised of 28 subunits and has a molecular weight of
700 kDa:
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Targeted Protein Degradation (8.3)
Next: Summary
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Summary (8.4)
Summary
●
●
●
Targeting of newly synthesized proteins is an integral component of
protein synthesis.
In prokaryotes, targeting is usually achieved by an N-terminal signal
sequence of about 20 mostly hydrophobic amino acids.
In eukaryotes, targeting is more complex due to the large number of
different cellular compartments:
Nuclear targeting via the nuclear pore using a nuclear
localization signal.
ER targeting (secretory pathway) via N-terminal signal
sequences using SRPs with subsequent attachment to the ER,
Followed by transport to the Golgi complex
❍
❍
❍
●
Protein degradation of damaged or obsolete proteins is carried out
by lysosomes, vesicles filled with degradating enzymes
in a ubiqutin-dependent process by specific proteases in a large
cytosolic complex called the proteasome.
❍
❍
Last Lecture: Expression Systems for Recombinant Proteins
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Final Exam (9.0)
Final Exam, BS99A
Final Exam, BS99B
●
●
●
●
●
●
●
There will be assigned seating (charts posted at PSLH doors at 10:00
am).
Bring your student ID.
Bring a pencil.
No calculators, no pagers, no hats, etc...
Multiple choice AND essay questions will cover lectures since the
mid-term.
After 12:15pm, please don't get up until all exams have been collected.
Questions regarding grading should be directed in writing and with a
detailed explanation to the TA.
Please fill out teaching evaluations (for my section only) and
turn them in after today's lecture.
Last Lecture: Expression Systems for Recombinant Proteins
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Final Exam (9.0)
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Expression Systems for Recombinant Proteins (9.1)
Lecture 9
Expression Systems for Recombinant Proteins
Recombinant protein expression is the
foundation of today's biomolecular research and
the thriving Biotech industry.
Goal: Overproduction of proteins for structural
& functional studies and for medical
& industrial applications.
As a 199 research student chances are high that
you will engage in some aspect of recombinant
protein expression.
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Expression Systems for Recombinant Proteins (9.1)
These techniques rely on:
●
the universality of the Genetic Code
●
knowing the Genetic Code
●
the relative similarity of the translational machinery (ribosome)
●
the rapid progress in molecular biology/genetic engineering over the past few
decades:
❍ sequence-specific nucleic acid hybridization (1961)
❍ sequence-specific cleavage of DNA (1962)
❍ DNA cloning/amplification (early 70s)
❍ DNA sequencing (mid 70s)
❍ Cutting and pasting pieces of DNA from one source into another:
■ excise with sequence-specific restriction endonuclease (more than 100
known, normally employed for defense by bacteria)
■ hybridize sticky ends
■ reform covalent phosphodiester bonds with DNA ligase (1967)
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Expression Systems for Recombinant Proteins (9.1)
Examples of Recombinant Protein Products
●
●
●
Hormones
❍ Insulin: Diabetes
❍ Human thyroid stimulating hormone
Blood clotting factors
❍ Coagulation factor VIII : hemophilia A.
❍ Coagulation factor IX: hemophilia B.
Interferons
❍ interferon-(alpha)-2a: chronic hepatitis C.
❍ gamma interferon: hepatitis B, C, herpes and viral enteritis.
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Expression Systems for Recombinant Proteins (9.1)
●
●
Immunization agents
❍ Hepatitis B vaccine: a non-infectious vaccine derived from Hepatitis B
surface antigen (HBSA) produced in yeast cells.
Research enzymes
❍ Restriction endonucleases
❍ Endoglycosidases: PNGase F
Next: DNA Cloning
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DNA Cloning (9.2)
DNA Cloning
Cloning:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Cell fractionation by conventional chromatographic methods to yield
about a microgram of protein.
The protein is analyzed to yield the identity of the first 30 amino acids
- its N-terminal amino acid sequence.
The Genetic Code is used to predict nucleotide sequences
corresponding to this amino acid sequence.
DNA fragments complementary to these sequences (oligomers of 1520 bases) are chemically synthesized.
The DNA fragments are then hybridized (base-paired) with total
cellular mRNA.
Long cDNA segments are produced from mRNAs complementary to
the DNA fragments using the enzyme reverse transcriptase.
Large amounts of this cDNA is obtained by cloning into plasmids
(amplification).
Selection of the right clone (several steps).
Finally, the cloned cDNA is incorporated into an expression vector or
plasmid and transferred into bacterial or yeast cells.
This is the starting point for scaled-up production of large amounts of
the protein.
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DNA Cloning (9.2)
Expression vectors or plasmids must contain:
●
●
●
●
origin of replication: DNA polymerase
selectable marker(s): antibiotic resistance
promoter: recognized by RNA polymerase
multiple clonig sites (restriction enzyme sites): cutting/pasting of DNA fragments
Plasmids are small circular molecules of extrachromosomal, double-stranded DNA. They
occur naturally in both bacteria and yeast where they replicate as independent units.
Unlike chromosomal DNA, plasmids usually occur as multiple copies.
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DNA Cloning (9.2)
Restriction endonuclease EcoRI cuts double-stranded DNA and generates sticky ends.
Sticky ends hybridize (base-pair) to each other and DNA ligase reforms covalent
phosphodiester backbone.
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DNA Cloning (9.2)
A piece of DNA can be inserted into a plasmid if both the circular plasmid and the source
of DNA have recognition sites for the same restriction endonuclease.
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DNA Cloning (9.2)
General scheme for insertion of gene of interest into an expression plasmid.
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DNA Cloning (9.2)
Next: Expression Systems
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Expression Systems (9.3)
Expression Systems
The DNA on the plasmid is transcribed to
mRNA which in turn is translated to protein.
Types of expression systems:
●
●
●
●
●
Bacterial: plasmids, phages
Yeast: expression vectors: plasmids, yeast artifical chromosomes (YACs)
Insect cells: baculovirus, plasmids
Frog oocytes: injected mRNA
Mammalian:
❍ viral expression vectors (gene therapy):
■ SV40
■ vaccinia virus
■ adenovirus
■ retrovirus
❍ Stable cell lines (CHO, HEK293)
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Expression Systems (9.3)
Bacterial expression systems:
●
Usually E. coli
●
A specific gene on a plasmid can produce 1-30% of the total protein.
●
●
●
For native protein:
❍ Ligate gene to bacterial promoter on plasmid.
❍ Watch for frame shift!
For fusion protein:
❍ DNA for gene of interest is inserted after the 3' or before the 5' terminus of
"carrier" gene (GST, GFP).
❍ Watch for frame shift!
❍ Advantages over native protein expression:
■ synthesized at high levels like a normal bacterial gene
■ often results in more stable product than native protein
■ fusion protein is generally larger than most E. coli proteins: easy
identification & purification
■ exploit functional features of carrier protein in purification
■ Caveat: often low yields when cleaving carrier from target protein
with protease
For His-tagged protein:
❍ By mutation introduce multiple histidine codons (6 or more) at the N-terminus
or C-terminus
❍ Purification over Nickel column: only proteins with poly-His tag will bind
tightly
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Expression Systems (9.3)
Common problems with bacterial expression systems:
●
●
●
●
●
Low expression levels:
❍ change promoter
❍ change plasmid
❍ change cell type
❍ add rare tRNAs for rare codons on second plasmid
Severe protein degradation:
❍ use proteasome inhibitors and other protease inhibitors
❍ try induction at lower temperature
Missing post-translational modification: co-express with kinases etc.
Glycosylation will not be carried out:
❍ use yeast or mammalian expression system
Misfolded protein (inclusion bodies):
❍ co-express with GroEL, a chaperone
❍ try refolding buffers
Next: Summary
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Summary (9.4)
Summary
●
●
●
●
Recombinant protein expression relies on the universality of the Genetic Code
It employs a host of sophisticated techniques to analyze, manipulate and copy DNA
(genetic engineering)
The gene of interest (DNA) is cloned into an expression plasmid or vector
The DNA on the plasmid is transcribed to mRNA which in turn is translated to
protein
Next: Final Exam
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http://bass.bio.uci.edu/~hudel/bs99a/final.html
Final Exam for BS99A
Final Exam Key for Dr. Osborne's section
Final Exam Key for the section on Protein Synthesis
Final Grades
The final exam will only cover lectures 11 to 28. It will
contribute up to 60 out of a total of 100 points towards the final
grade.
The only valid excuse for missing this exam is a substantiated
medical emergency or written arrangements made with Dr.
Luecke. Requests and reasons for missing the exam must be
submitted in writing. Makeup exams will be oral and, unlike the
written exam, comprehensive!
Questions regarding grading need to be directed before noon
on June 30 in writing and with a detailed explanation to one of
the TAs. Only if no satisfactory solution can be found with the
TAs, a written request (email or paper) with a detailed
explanation regarding the circumstances should be sent to Dr.
Luecke.
Dr. Hartmut Luecke
3205 McGaugh Hall
ZOT 3900
http://bass.bio.uci.edu/~hudel/bs99a/final.html5/24/2007 12:58:08 PM
Bio 99A 2005
Key Osborne
Answers: Version1/Version 2
1/14 ). (1 point) Complete the following sentence with 3-5 words
The alpha (α) subunit of E.coli RNA polymerase functions in transcription by …
binding upstream DNA non-sepcifically and binding to other regulatory proteins (like
CAP)______
+
2/15). (1 point) In the regulation for the Lac operon of E. coli, the wild type I is recessive to what two
important types of lac operon regulatory mutations (use no more than two words )
IS and Oc
3/16 ). (total of 3 points, one-two sentences of no more than 25-30 total words, no partial credit).
In the TRP operon of E. coli, if there was an insertion mutation in the leader DNA that resulted in an
additional 40 random nucleotides inserted into the leader RNA sequence between stem region 1 and
stem region 2, how would attenuation likely be affected when intracellular Tryptophan levels are high
(would attenuation be increased or decreased-1 point) and why ? (2 points))
attenuation would decrease, The insertion means that when the ribosome does not stall, it cannot
cover region 2 effectively so it is free to more frequently pair with region 3.
4/17 ). (1 point, one to three words) RNA polymerase III transcribes which major class of RNA in
eukaryotes? tRNA
5/18 ). (1 point-no partial credit) When lactose is added to a bacterial culture of E. coli with each of
the following genotypes, what would be the pattern for β-galactosidase expression for each haploid
strain below (a and b below) note that the solid line on the answer sheet provides the pattern for βgalactosidase enzyme activity in a strain that is completely wild type (I+ P+O+Z+Y+)
-
a). I P+O+Z+Y+
-
b). IS P+OcZ Y+
Copyright © 2005
Unless otherwise indicated, all materials on these pages are copyrighted by Drs.Timothy F Osborne and Hartmut Luecke. All rights reserved. No part of
these pages, either text or image may be used for any purpose other than personal use. Therefore, reproduction, modification, storage in a retrieval system or
retransmission, in any form or by any means, electronic, mechanical or otherwise, for reasons other than personal use, is strictly prohibited without prior
written permission.
Bio 99A 2005
Key Osborne
6/19). (1 point, one word) When the two above strains are combined to form a partial diploid strain
-
containing both Lac operons, what is the phenotype (Lac + or Lac )
-
Lac
7/20). (2 points, no partial credit) If the TRP regulatory gene (TRP R) encoded a protein that
stimulated RNA polymerase binding to the promoter (positive control mechanism) and yet the operon
was still repressed by the accumulation of tryptophan what would be the most likely effect of
tryptophan on the DNA binding properties of the TRP R regulatory protein?
TRP would decrease TRPR binding to operator
8/21 ). (one point for each-total of 3 points) As the mechanism of attenuation was described in class,
there are three molecular features that are absolutely required. List them
1).__Coupled transcription (RNA synthesis) and translation (protein synthesis_
2).__Alternative secondary structures in leader mRNA___
3).__Clustered (2 or more) codons for the regulatory amino acid in the leader
mRNA_
9/22 ). (2 points-no partial credit) In the binding of the Lac I protein to the DNA, we discussed a
general equlibrium based method for evaluating proteins binding to DNA. These same principles apply
to RNA polymerase (RNAP) from E. coli binding to different promoters. If the solid line on the graph
provided on the answer sheet describes the binding of RNA polymerase to a promoter that is identical to
the “consensus sequence”, insert a graph that would describe RNA polymerase binding to a weak
promoter such as from the Lac operon –use a dashed line graph?
10/21). (1 point) The core E. coli RNA polymerase contains which subunits and how many copies of
each? 2-alpha, 1-beta, 1-beta prime
Copyright © 2005
Unless otherwise indicated, all materials on these pages are copyrighted by Drs.Timothy F Osborne and Hartmut Luecke. All rights reserved. No part of
these pages, either text or image may be used for any purpose other than personal use. Therefore, reproduction, modification, storage in a retrieval system or
retransmission, in any form or by any means, electronic, mechanical or otherwise, for reasons other than personal use, is strictly prohibited without prior
written permission.
Bio 99A 2005
Key Osborne
11/22 ). (1 point) What is the fundamental difference between RNA polymerase and DNA polymerase
(besides one being involved in DNA replication and one being involved in transcription). ? One sentence
of up to 15-16 words and be sure to refer to each polymerase in your answer. (1 point)
_RNAP can initiate de nove0. DNAP require a primer with 3’OH for extension
12/23 ). (1 point) In protein assembly onto a core eukaryotic promoter for RNA polymerase II (on to
the TATA box) which proteins are bound at the promoter before RNA polymerase is recruited?
TF IID, A and B (note TBP is only part of TFIID)
13/24 ). Consider a strain of E. coli with the following genotype for the Lac operon (I- P+OCZ+Y+)
A). (1 point) use a dashed line graph to draw the pattern of β-galactosidase expression over time
when only Lactose is added (the solid line describes what would happen to the wild type strain (I+
P+O+Z+Y+)
B). (1 point) use a dashed line graph to draw the pattern for β-galactosidase expression over time
when lactose and a high concentration of glucose are added together (the solid line describes what
would happen to the wild type strain when only lactose is added (I+ P+O+Z+Y+)
C). (3 points) Based on what we discussed in class, explain the difference between A and B above
(one to two sentences total of 25 words)
C). at high glucose, cAMP levels are low and CAP does not bind to its DNA site and the weak lac
promoter is not expressed
14/25). (1 point, up to three words) Which specific eukaryotic RNA polymerase is inhibited only
when very high concentrations of the drug alpha (α) amanitin are added to a transcription reaction?
RNA Polymerase I
Copyright © 2005
Unless otherwise indicated, all materials on these pages are copyrighted by Drs.Timothy F Osborne and Hartmut Luecke. All rights reserved. No part of
these pages, either text or image may be used for any purpose other than personal use. Therefore, reproduction, modification, storage in a retrieval system or
retransmission, in any form or by any means, electronic, mechanical or otherwise, for reasons other than personal use, is strictly prohibited without prior
written permission.
Bio 99A 2005
Key Osborne
15/26 ). (1 point, one word) In mammalian cells, the type of chromatin associated actively transcribed
genes is called
euchromatin
16/27 ). (1 point, one sentence, 10-12 words) What is the role of TF IIA in transcription?
Stabilize TBP bound to TATA box (not specific enough to say just stabilize TBP)
17/28 ). (1 point, one to two words) If DNA was wrapped around each nucleosome three times, how
many total basepairs of DNA would be associated with each nucleosome?
18/31). (3 points, two sentences, 20-30 words) On the planet of Tatuine, the Empire Research
Corporation has found a new life force that threatens the stability of the Empire. When researchers
evaluate RNA expression from this new organism they perform a RNA synthesis experiment very
similar to what we described in Bio 99. When RNA was synthesized in the absence or presence of
different concentrations of the inhibitor alpha (α) amanitin the results were analyzed by gel
electrophoresis as displayed in the figure below. What can you conclude about the RNA synthesizing
enzymes of this new life force?
Two a-amanitin sensitive enzymes, one transcribes tRNAs and rRNAs and is inhibited by low
drug concentration and a second enzyme that transcribes mRNAs and is inhibited by only a high
alpha-amanitin level
Copyright © 2005
Unless otherwise indicated, all materials on these pages are copyrighted by Drs.Timothy F Osborne and Hartmut Luecke. All rights reserved. No part of
these pages, either text or image may be used for any purpose other than personal use. Therefore, reproduction, modification, storage in a retrieval system or
retransmission, in any form or by any means, electronic, mechanical or otherwise, for reasons other than personal use, is strictly prohibited without prior
written permission.