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Translation of RNA
The Genetic Code and Protein Metabolism
The Process of Protein Synthesis
The Three Stages of Protein Synthesis
Post-translational Processing of Protein
Regulation of Protein Synthesis
1
Characteristics of
protein synthesis
Occurs on a ribosome - a complex of
ribosomal RNA and proteins.
Made of two subunits
• large - three rRNA and about 49 proteins
• small - one rRNA and about 33 proteins
• Together they provide a platform for
synthesis
You can think of a ribosome like a tape
player, where the tape is mRNA.
2
Ribosomes
Two ribosomal subunits join to form a polysome.
large subunit
small subunit
synthesis
platform
3
Where protein synthesis begins
It has been verified that protein synthesis
begins at the amino terminus.
R'
R
growing chain
N
N
H
C
H
COO -
R
O
C
H
C
+
H2N
C
H
COO -
R'
growing chain
N
N
H
N
H
C
H
COO 4
Role of tRNA
Transfer RNA
•
Serves as an adapter
molecule to translate
the 4 letter language of
mRNA into 20 amino
acids.
•
Binds to a single amino
acid at one end.
•
Serves to bring the
proper amino acid to the
right spot on the mRNAribosome complex.
5
tRNA
HO-
C
Site of amino
acid attachment
G
A
U
G
U
C
G
G U
Three base
anticodon site
A
C G
G
C
A
C
A
C
G
G
U
G
C
G
C
G
U
C
G
G
C
U
U
G
C
A
G
G
C
C
U
U
A
G
U
C
G C
C
C
G
G
C
G
C
U
G
C
G
U
A
C
G
C
G
C
G
A
U
G
U
G
C
G
C
G
Point of
attachment
to mRNA
6
Attachment of amino acids
Amino acids are linked to tRNAs by
aminoacyl-tRNA synthetases.
These enzymes are able to recognize both
the correct tRNA and amino acid.
amino acid + ATP
aminoacyl adenylate + PPi
aminoacyl adenylate + tRNA
PPi + H2O
aminoacyl-tRNA + AMP
2Pi
7
Attachment of an amino acid
amino acyl group
G
A
R
O
CH
C
HO-
U
G
C
G U
A
C G
G
C
ester bond
A
C
+
NH3
U
G
O
C
A
C
G
G
U
G
C
G
C
G
U
C
G
G
C
U
U
G
C
A
G
G
C
C
U
C
C
G
G
U
U
A
G
C
G C
C
G
C
U
G
C
G
U
A
C
G
C
G
C
G
U
A
G
G
U
G
C
G
C
8
Genetic code, mRNA
and protein amino acids
Important terms
Triplet
A set of three nucleotide bases on mRNA for one
amino acid.
Nonoverlapping
A set of three adjacent bases are treated as a
complete group - codon.
No punctuation.
There are no intervening bases between triplets.
9
Genetic code, mRNA
and protein amino acids
Important terms
Degenerate
A single amino acid may have more than one
triplet code. There is usually a sequential
relationship between these codes.
Universal
The same genetic code is used by all organisms
except mitochondria and some algae.
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Amino acid codons
alanine
GCA, GCC, GCG
GCU, AGA, AGG
arginine
AGA, AGG, CGA
CGC, CGG, CGU
asparagine AAC, AAU
aspartate GAC, GAU
cysteine
UGC, UGU
glutamate GAA, GAG
glutamine CAA, CAG
glycine
GAA, GCC, GGG
GGU
histidine
CAC, CAU
isoleucine AUA, AUC, AUU
leucine
CUA, CUC, CUG
CUU, UUA, UUG
lysine
AAA, AAG
methionine
AUG
phenylalanine UUC, UUU
proline
CCA, CCC
CCG, CCU
serine
UCA, UCC
UCG, UCU
AGC, AGU
threonine
ACA, ACC
ACG, ACU
tryptophan
UGG
tyrosine
UCA, UCU
valine
GUA, GUC
GUG, GUU
11
Three stages of protein synthesis
Production of a protein requires several
players.
aminoacyl - tRNA
source of amino acids
mRNA
the information source for construction of
the protein sequence
Ribosomes
platform for synthesis - rRNA and protein.
12
Steps in protein synthesis
Step One - Initiation
A special protein is required to bring the
ribosome parts and mRNA together.
It recognizes the initiation (START) codon
(AUG).
Once formed, the ribosome complex will
- hold the mRNA in place.
- provide binding sites for the growing
protein and incoming amino acids.
13
Steps in protein synthesis
Step Two - Chain elongation
Amino acid is added sequentially to the
peptide chain.
An enzyme, peptidyl transferase, is used
to move the ribosome down the mRNA
strand - translocation.
14
Steps in protein synthesis
Step Three - Termination
When one of three codons (UAA, UAG or UGA)
is encountered, there is no tRNA that matches.
Protein synthesis stops.
A releasing factor is attracted to the site.
This results in the growing protein being
released from the ribosome.
The ribosome complex then falls apart into the
original subcomplexes.
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Protein synthesis
Protein synthesis and energy
The energy requirements for synthesis are
quite high.
• Two anhydride bonds in ATP are cleaved
on activation of each amino acid and
synthesis of an aminoacyl-tRNA.
• One GTP is required for entry of each
amino acid into the ribosomal unit.
• One GTP is required during each
translocation step.
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Post-translational
processing of proteins
Protein synthesis establishes the primary
structure for a protein.
Additional processing is required to
convert it to it’s biologically active form.
This may include:
folding
chemical modification
attachment of other groups
18
Protein folding
Results from interaction of side chains.
Proteins called chaperones act as catalysts
to guide this process.
Possible side chain interactions:
• Similar solubilities
• Ionic attractions
• Attraction between + and - side chains
• Covalent bonding
19
Protein folding
Disulfide
Crosslink
Hydrophobic
interaction
-S-S-
-COO-
+
H3N -
Salt bridge
Hydrogen
bonding
20
Protein folding
Side chain interactions
Help maintain specific structure.
Oxidation of cystine - crosslink formation.
O
||
HO-C-CH-CH2-SH
|
NH2
oxidation
[O]
O
||
HS-CH2-CH-C-OH
|
NH2
covalent
disulfide
bond
O
O
||
||
HO-C-CH-CH2-S - S-CH2-CH-C-OH +H2O
|
|
NH2
NH2
21
Protein folding
An example of how a S-S
crosslink can affect folding
20 glycines - cysteine
40 glycines
crosslink
20 glycines - cysteine
 - helix structure
22
Biochemical modification
Proteolytic cleavage
About half of all proteins require the
removal of their N-terminus amino acid to
become active.
In addition, many enzymes are produced
in an inactive form - zymogens. They
require that one or several specific bonds
be cleaved to produce the active form.
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Biochemical modification
Amino acid modification
Phosphorylation
Serine, threonine and tyrosine residues
may be modified by transfer of a
phosphoryl group.
Hydroxylation
Proline and lysine may be converted to
hydroxyproline or hydroxylysine.
24
Biochemical modification
Attachment of carbohydrates
This process is used for the production of
glycoproteins.
Attachment of prosthetic groups.
The addition of small organic, inorganic
or organometallic groups - heme, FAD,
biotin, ...
25
Protein targeting
Most proteins are synthesized by the
ribosomes in the cytoplasm.
However, they may be required in other
cellular regions and organelles
Protein targeting deals with the process of
sorting out and moving proteins to where
they are needed.
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Protein targeting
• In general, proteins that must be
transported are produced with an extra
sequence of 15-36 amino acids - at the
amino terminus.
• The sequence marks it for transport.
• The sequence is removed by hydrolysis
upon arrival.
• Proteins sent to the nucleus use an internal
sequence that is not cleaved.
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Protein degradation
After a protein has served it’s purpose or
becomes damaged, it is marked for
destruction.
The turnover rate varies by protein.
Protein
Rat liver RNA polymerase I
Rat liver cytochrome c
Human hemoglobin
half-life
1.5 min
150 min
100 days
28
Protein degradation
Ubiquitin pathway
Important route for protein labeling and
degradation in eukaryotic cells.
Ubiquitin
• A small protein with 76 amino acid
residues.
• It is highly conserved.
• Yeast and human ubiquitin differ at only 3
of the 76 residues.
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Ubiquitin
30
Ubiquitin pathway
• Ubiquitin is covalently attached via a
peptide bond to lysine residues.
• Several ubiquitin molecules are often
attached to a single protein.
• Degradation then occurs via proteolytic
action.
O
ubiquitin
C
N

N
Lys
protein
H
C
31
Regulation of protein synthesis
• A typical bacterial cell has about 4000
genes in its DNA genome.
• The human genome has an estimated
100,000 to 150,000
• Only a small fraction of these genes is
used by a cell at any given time, if at all.
• The amount of each protein generated
must be carefully regulated to account for
the needs of the cell.
32
Regulation of gene expression
There are many steps that can be regulated.
• transcription
• post-transcriptional processing
• mRNA degradation
• protein synthesis
• post-translational processing
• protein degradation
Most gene expression is controlled at the
level of transcription initiation.
33
Regulation of gene expression
Nucleotides
mRNA
degradation
protein
degradation
Amino
acids
posttranslational
processing
transcription
DNA
posttranscriptional
processing
primary
transcript
mature
RNA
translation
inactive
protein
Active
protein
34
Regulation of gene expression
Two fundamental types of gene expression.
Expression of constitutive genes.
Continuous transcription which produces a
constant level of certain proteins.
Expression of inducible or repressible genes.
Genes that can be activated (induced) or
deactivated (repressed). This allows for
varied levels of RNA. They are regulated by
RNA polymers and molecular signals.
35
Principles of regulating
gene expression
In many prokaryotic cells, genes for proteins
of related function are clustered in units.
The components in these units are:
• Structural genes - the gene which is to be
transcribed & translated.
• Promoter region - responsible for RNA polymerase
binding to the initiation site.
• Binding site for inducers.
• Binding site for repressors - called operators.
All structural genes are regulated by nucleotide
sequences upstream from the start site (+1).
36
Principles of regulating
gene expression
RNA polymerase
• The key participant in transcription.
• It initiates transcription by binding to a
DNA promoter region.
• The sequences in each promoter region
determines the affinity for its binding.
• For inducible or repressible genes, other
levels of control are superimposed regulatory proteins and other signals.
37
Principles of regulating
gene expression
Eukaryotic gene regulation is a more
complicated process.
• Complex sets of regulatory elements are
present in promoter regions.
• Three classes of RNA polymerase using
different modes of regulation are present.
• The DNA is much more complex in both
size and structure.
38
Principles of regulating
gene expression
RNA polymerase activity is mediated by
regulatory proteins of two major types.
Activators. Bind to promoter regions and
assist the binding of RNA polymerase to
the adjacent promoter.
Repressors. Bind to specific base
sequences in the promoter region and
prevent RNA polymerase in gaining
access.
39
Principles of regulating
gene expression
Most regulatory process can be classified
into one of four mechanistic types.
Positive regulation
Transcribes until a molecular signal is sent.
Transcribes after a signal is sent.
Negative regulation
Blocks transcription until a signal is sent.
Stops transcription when the signal is sent.
40
Positive regulation
activator RNA polymerase
molecular
signal
Binding of
molecular
signal
causes
dissociation of
activator from DNA
Binding of
molecular
signal
causes strong
binding of
of activator to DNA
41
Negative regulation
operator
Binding of
the molecular
recognition
signal causes
dissociation
of the operator
from the DNA
Binding of
the molecular
recognition
signal causes
stronger
binding of the
operator to DNA
42
Principles of regulating
gene expression
Regulatory proteins have common, discrete
binding domains.
• 20-100 amino acid residues.
• They bind because the domain is an exact
fit for the outer edge of the DNA helix.
• Held together by hydrogen bonding that is
not disruptive to DNA.
• Lysine, arginine, glutamate, asparagine
and glutamine form the hydrogen bonds.
43
Classes of regulatory proteins
Helix-turn-helix motif
About 20 amino acid residues, in two
helical regions and a turn.
44
Classes of regulatory proteins
Zinc finger motif
Only found in
eukaryotic
regulatory
proteins.
Cys
His
Cys
Zn
His
45
Classes of regulatory proteins
Leucine zipper motif
Features an -helix region of approximately 30
residues. Leucine occurs as every seventh one.
This allows two molecules of the protein to form
a zipper like region.
46