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Chapter 17
From Gene to Protein
Overview: The Flow of Genetic Information
• the information content of DNA is in the form of specific sequences
of nucleotides
• the DNA inherited by an organism leads to specific traits by
dictating the synthesis of proteins
• proteins are the links between genotype and phenotype
• Gene expression = process by which DNA directs protein synthesis
– includes two stages: transcription and translation
Basic Principles of Transcription and Translation
• RNA is the bridge between genes and the proteins for which they
code
• Transcription = synthesis of RNA
– using information in DNA
• Translation = synthesis of a polypeptide
– using information in the mRNA
– Ribosomes - sites of translation
DNA
RNA
Protein
The Products of Gene Expression: A Developing Story
•
original hypothesis posed by scientists: one gene – one
enzyme
• BUT a lot of proteins aren’t enzymes - researchers later
revised the hypothesis: one gene–one protein
• many proteins are composed of several polypeptides
– each of which has its own gene
• can now restated the hypothesis as the one gene–one
polypeptide hypothesis
• **Note: common to refer to gene products as proteins rather
than polypeptides
DNA vs. RNA
Types of RNA
 mRNA = messenger RNA
– majority of RNA found in a cell
– carries the genetic information which will be translated into a
protein sequence
– defined by the presence of a “cap” at its 5’ end and a long tail of
adenines at its 3’ end = “poly-A tail”
Types of RNA

rRNA = ribosomal RNA



found in the nucleolus
combines together with the large and small ribosomal subunits to
form the functional ribosome (protein translation)
rRNA is transcribed in the nucleolus by RNA polymerase I
28S rRNA
Types of RNA

tRNA = transfer RNA



actually translates the message coded in the mRNA into a protein
sequence which will become a function protein
tRNA is transcribed in the nucleoplasm by an enzyme called RNA
polymerase III
then exported into the cytoplasm where AA are added
5’
3’
3’
5’
-transcription of RNA is similar to DNA replication – RNA is made in the 5’ to 3’
direction
-enzyme called an RNA polymerase binds to only one of the DNA strands = the
anti-sense (template strand)
-it moves along the template DNA strand (in the 3’ to 5’ direction) and reads the
nucleotide and adds a complementary RNA base
- a growing strand of RNA complementary to the DNA strand results
-BUT rather than a T being paired with an A – U becomes the partner to A
Transcription
-a human gene is also known as a transcription unit = stretch of DNA that is transcribed
into RNA
-a transcription units is comprised of:
1. coding sequence – gives rise to protein strand upon translation
-contains regions of code = “exons” – code for amino acids
-and regions of junk = “introns” – spliced out in the nucleus
5’
3’
Exon
Intron
Exon
Intron
Exon
Intron
Exon
Transcription
-
2. untranslated regions (UTRs) - the regions upstream and downstream of the
coding region that are transcribed but NOT translated into a protein
- -play an important role in translation – can influence the binding of the ribosome
to the mRNA
- -also play a role in exporting the mRNA into the cytoplasm
Transcription
• genes are also associated with additional sequences of DNA
1. core promoter sequence – for the binding of the RNA polymerase
-RNA polymerase recognizes specific sequences of nt’s
-binding is helped out by transcription factors
2. enhancer regions – help enhance transcription
can be several thousands of base pairs upstream of the gene
Transcription
•
•
•
•
the transcription unit is transcribed by an RNA polymerase
three types of RNA polymerase – I, II and III
RNA polymerases create an RNA strand called a primary transcript
• must be modified to produce the final mRNA, tRNA or rRNA
RNA polymerase II transcribes protein coding genes into a primary transcript called premRNA – this is then is processed into mRNA
– genes for tRNA are transcribed in the cytoplasm by RNA polymerase III – primary
transcript is modified into tRNA
– genes for rRNA is transcribed in the nucleolus by RNA polymerase I – primary
transcript is modified into rRNA
-3D representation of the
RNA polymerase II enzyme
Transcription
• three stages of transcription
– Initiation: binding of the RNA polymerase to the
promoter
• special sequences denote this region
– Elongation: movement of the RNA polymerase along
the anti-sense DNA strand and synthesis of the RNA
transcript
– Termination: release of the RNA polymerase from
the DNA
• special sequences denote this region
• differs between prokaryotes and eukaryotes
Promoter
Transcription unit
5
3
Start point
RNA polymerase
DNA
3
5
1. Initiation – RNA polymerase binds to a special sequence
of nucleotides called the promoter
-certain sections of the promoter are important in
1 A eukaryotic promoter
polymerase binding = core promoter
Promoter
Nontemplate strand
DNA
-in prokaryotes the promoter binds the RNA
5
3
5
3
polymerase without help
TATA box
Template strand
Start point
2 Several transcription
-in eukaryotes – the polymerase requires the assistance
Transcription
factors bind to DNA
factors
of proteins called transcription factors
-specific transcription factors bind to the promoter
5
3
3
5
first and then help position the polymerase at the
3 Transcription initiation
promoter
complex forms
-additional transcription factors then bind
RNA polymerase II
Transcription factors
-entire complex is called the Transcription Initiation
5
3
Complex
3
5
3
TAT AAAA
AT AT T T T
5
RNA transcript
Transcription initiation complex
sequence given in texts is that of the sense strand
Promoter
Transcription unit
5
3
Start point
RNA polymerase
DNA
3
5
1 Initiation
Nontemplate strand of DNA
3
5
5
3
Unwound
DNA
RNA
transcript
Template strand of DNA
1. Initiation cont…
-RNA polymerase unwinds the DNA helix (acts as a helicase) –
exposes about 10 to 20 nucleotides for copying
-RNA polymerase holds the DNA helix open (acts like the SSBs)
-RNA polymerase initiates RNA synthesis without the need for a
primer
Promoter
Transcription unit
5
3
Start point
RNA polymerase
3
5
DNA
1 Initiation
Nontemplate strand of DNA
3
5
5
3
Unwound
DNA
RNA
transcript
2. Elongation – RNA polymerase synthesizes
a complementary RNA strand
-RNA primary transcript grows in the 5’ to 3’
direction
-uses uracil instead of thymine
-the DNA strands reform their helix once the
RNA polymerase moves past the area
-the mRNA strand emerges from the
polymerase-DNA complex
Template strand of DNA
2 Elongation
Rewound
DNA
5
3
3
5
Nontemplate
strand of DNA
RNA nucleotides
3
5
RNA
transcript
RNA
polymerase
A T C C A A
3
C
5
3 end
C A U C C A
5
Multiple RNA polymerases per DNA template
5
T A G G T T
Direction of transcription
Template
strand of DNA
Newly made
RNA
3
Promoter
Transcription unit
5
3
Start point
RNA polymerase
3
5
DNA
1 Initiation
Nontemplate strand of DNA
3
5
5
3
Unwound
DNA
RNA
transcript
Template strand of DNA
2 Elongation
Rewound
DNA
5
3
3
5
3
5
RNA
transcript
3 Termination
3
5
5
3
5
Completed RNA transcript
3
Direction of transcription (“downstream”)
3. Termination – RNA polymerase
reaches a specific sequence of
nucleotides and stops
transcription
-the RNA polymerase detaches
from the DNA
-the pre-RNA primary transcript is
released
-in prokaryotes – a termination
sequence that detaches the
polymerase
-in eukaryotes – the RNA
polymerase transcribes a
sequence called a polyadenylation signal
– for the release of the pre-RNA
from the polymerase
Transcription
• to modify the primary transcript into mRNA – the
following modifications are made:
– a 5’methylated cap is added to the 5’end
– addition of a 3’ poly A tail
– the coding sequence is “edited” = splicing
Eukaryotic cells modify RNA after transcription
• enzymes in the eukaryotic nucleus modify pre-mRNA before
exporting the mRNA to the cytoplasm
– known as RNA processing
• 5’ methylated cap – plays a role in the docking of the ribosome
to mRNA – for translation
– modified guanine nucleotide added after the transcription of about 20 to 40
nucleotides
5
G
Protein-coding
segment
P P P
5 Cap 5 UTR
Polyadenylation
signal
AAUAAA
Start
codon
Stop
codon
3 UTR
3
AAA … AAA
Poly-A tail
Eukaryotic cells modify RNA after transcription
• 3’ poly A tail – plays a role in the export of the mRNA
into the cytoplasm
– after transcription – an enzyme adds 20 to 250 adenine nucleotides after
the poly-adenylation signal sequence
– also prevents degradation of the mRNA once its in the cytoplasm
5
G
Protein-coding
segment
P P P
5 Cap 5 UTR
Polyadenylation
signal
AAUAAA
Start
codon
Stop
codon
3 UTR
3
AAA … AAA
Poly-A tail
RNA Splicing
•
most eukaryotic genes and pre-RNA transcripts have long noncoding stretches of
nucleotides that lie between coding regions
– the noncoding regions are called intervening sequences, or introns
– coding regions are called exons because they are eventually expressed in the form of a
protein
– RNA splicing removes introns and joins exons, creating an mRNA molecule
with a continuous coding sequence
– the way you splice can also create multiple isoforms from one RNA transcript
5 Exon Intron Exon
Pre-mRNA 5 Cap
Codon
130
31104
numbers
Intron
Exon 3
Poly-A tail
105
146
Introns cut out and
exons spliced together
mRNA 5 Cap
Poly-A tail
1146
5 UTR
Coding
segment
3 UTR
• RNA splicing is carried out by spliceosomes
• Spliceosomes = several proteins and small nuclear
ribonucleoproteins (snRNPs) that recognize specific sequences
found in introns called splice sites
•
snRNPs – found in the nucleus and are made of small nuclear RNA
(snRNA) and proteins
RNA transcript (pre-mRNA)
5
Exon 1
Protein
snRNA
Intron
Exon 2
Other
proteins
snRNPs
RNA transcript (pre-mRNA)
5
Exon 1
Intron
Protein
snRNA
Exon 2
Other
proteins
snRNPs
1. snRNPs and other proteins
combine to form the spliceosome
Spliceosome
5
2. the spliceosome brings the ends
of two exons together
-forms a “lariat” out of the intron
Spliceosome
components
3. the spliceosome cuts the
pre-mRNA and releases the intron
for degradation
5
mRNA
Exon 1
Exon 2
Cut-out
intron
RNA Splicing
•
genes can encode for more than one
protein
–
•
•
DNA
depending on what segments of RNA are
treated as exons and what are treated as
introns during splicing
Exon 1
–
Exon 2
Intron
Exon 3
RNA processing
Translation
Domain 3
cut out a domain – get a different protein
also - exon shuffling may result in the
evolution of new proteins
–
Intron
Transcription
so the way you splice can determine
what proteins eventually get made =
alternative RNA splicing
proteins often are composed of discrete
regions called domains – coded for by
distinct exons
–
•
Gene
introns increase the probability of crossingover between alleles
creates new exon combinations
Domain 2
Domain 1
Polypeptide
Splicing
• for an animation go to
http://sumanasinc.com/webcontent/animatio
ns/content/mRNAsplicing.html
• (don’t worry about the actual proteins!)
Translation
• process of converting an mRNA message into a strand of amino acids that will be
processed into a mature functional protein
• performed by the ribosome in combination with tRNA molecules
• prokaryotes - translation of mRNA can begin before transcription has finished – no
separation between the mRNA and the ribosome
• eukaryotic cell- the nuclear envelope separates transcription from translation
–
mRNA has to be exported out of the nucleus first
DNA
template
strand
5
3
A
C
C
A
A
A
C
C
G
A
G
T
T
G
G
T
T
T
G
G
C
T
C
A
3
5
DNA
molecule
Gene 1
TRANSCRIPTION
Gene 2
U
mRNA
G
G
U
U
U
G
G
C
U
C
5
A
3
Codon
TRANSLATION
Protein
Trp
Phe
Gly
Ser
Gene 3
Amino acid
•
•
•
•
–
•
61 amino acid codons; 3 stop codons
the code is redundant - each amino acid
can be coded for by more than one codon
• e.g. alanine – GCU, GCC, GCA and GCG
• the GC defines the amino acid as alanine
•
in many cases the 3rd codon is important in
defining the amino acid
–
serine –codons are: AGU, AGC
– BUT arginine codons are: AGA and AGG
The Genetic Code
1964
Second mRNA base
A
C
U
UUU
Phe
U
UUC
UAU
UCU
UGU
Tyr
UCC
U
UAC
UGC
C
UCA
UAA Stop
UGA Stop
A
UUG
UCG
UAG Stop
UGG
Trp
G
CUU
CCU
CAU
CGU
Leu
His
A
Cys
Ser
UUA
C
G
CUC
Leu
CAC
CCC
CCA
CUG
CCG
CAG
AUU
ACU
AAU
AUC
Ile
AUG
CAA
Met or
start
GUU
Gln
AAC
ACC
ACA
AAA
ACG
AAG
GCU
GAU
CGA
C
Arg
CGG
Asn
Thr
AUA
CGC
Pro
CUA
U
AGU
G
Ser
AGA
U
C
AGC
Lys
A
Arg
A
AGG
G
GGU
U
Asp
G
GUC
GUA
GUG
GCC
Val
GCA
GCG
GAA
GAG
C
GGC
GAC
Ala
Glu
GGA
GGG
Gly
A
G
Third mRNA base (3 end of codon)
•
How are the instructions for assembling
amino acids into proteins encoded into
DNA?
20 amino acids - only four nucleotide bases
in DNA
how many nucleotides correspond to an
amino acid?
the mRNA nucleotide sequence is “read” in
groups of 3 nucleotides = “codons”
each codon codes for 1 of the 20 amino
acids that make up proteins
called the “genetic code”
First mRNA base (5 end of codon)
•
Molecular Components of Translation
• two components
• 1. transfer RNA (tRNA)
• 2. the ribosome
tRNA
•
•
•
•
tRNA molecule consists of a single RNA strand that is only about 80 nucleotides
long
at one end – anticodon site for the hybridization with the mRNA template
at the other end – attachment site for the amino acid that corresponds to the
mRNA codon
transcribed in the cytoplasm by RNA polymerase III – it folds into its
characteristic shape spontaneously due
to regions that complement each other
3
Amino acid
attachment
site
5
Amino acid
attachment
site
5
3
Hydrogen
bonds
Hydrogen
bonds
A A G
3
Anticodon
(a) Two-dimensional structure
Anticodon
(b) Three-dimensional structure
5
Anticodon
(c) Symbol used
in this book
Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P Adenosine
P P P Adenosine
P Pi
ATP
Pi
-amino acids are attached in
the cytoplasm by enzymes
called
aminoacyl-tRNA –synthetases
-one end fits the amino acid,
the other end fits the tRNA
-20 synthetases – each is specific
for only one kind of tRNA
-the tRNA attached to an AA is
called a ‘charged tRNA’
Pi
tRNA
Aminoacyl-tRNA
synthetase
tRNA
Aminoacyl tRNA
(“charged tRNA”)
Amino
acid
P Adenosine
AMP
Computer model
tRNA and the 3rd codon “wobble”
• the tRNA recognizes the codon “triplet” on the mRNA
template
• attached to the tRNA is the amino acid corresponding
to this codon
• there are 61 amino acid codons – so there should be
61 tRNAs
• there are only 45 tRNAs
– some tRNAs can bind more than one codon
• the rules for complementary base pairing at the third
NT of the codon are less stringent
– “flexible” base pairing at this NT = Third Codon Wobble
Ribosomes
• machine of translation
• made in the nucleolus in eukaryotic cells
• comprised of two subunits of proteins (large and small) linked
together with a piece of rRNA
– eukaryotes: 40S small subunit = 33 proteins + 18S rRNA
+ 60S large subunit = 50 proteins + 28S rRNA (+ 5.6S rRNA + 5S rRNA)
– rRNA is transcribed in the nucleolus, proteins are imported from cytoplasm
– everything is assembled in the nucleolus
– subunits are exported out via nuclear pores
– prokaryotic ribosomes and similar but smaller
Ribosomes
• within the large subunit are two sites for the binding of tRNA
– P-site or Peptidyl-tRNA site – “old” AA
– A-site or aminoacyl-tRNA site – incoming AA
• and one E site/Exit site for the exit of the tRNA off the
ribosome
P site (Peptidyl-tRNA
binding site)
Exit tunnel
A site (AminoacyltRNA binding site)
E site
(Exit site)
E
mRNA
binding site
P
A
Large
subunit
Small
subunit
Ribosomes
• eukaryotic ribosomes are similar but are larger vs. prokaryotes
• most evidence now identifies the rRNA as being the catalyst for
the formation of the peptide bond and the growth of the
polypeptide chain
– RNA with enzymatic activity = ribozyme
Growing polypeptide
Amino end
Next amino
acid to be
added to
polypeptide
chain
E
tRNA
mRNA
5
3
Codons
(c) Schematic model with mRNA and tRNA
Building a Polypeptide
• 3 stages of translation:
– Initiation
– Elongation
– Termination
• all three stages require protein “factors”
– called initiation factors or IFs
– in eukaryotes – known as eIFs
1. Initiation of Translation
• the small subunit of the ribosome binds onto the mRNA sequence near the 5’ methylated
cap
• this subunit already has an initiator tRNA (bound to methionine) associated with it
• binding of the small subunit is helped by numerous eukaryotic initiation factors (eIFs)
• the small subunit then glides down the mRNA “scanning” for the first codon - START codon
= AUG (methionine)
-stops so that initiator tRNA can hybridize with the start codon
Large
ribosomal
subunit
3 U A C 5
5 A U G 3
P site
P i

Initiator
tRNA
mRNA
GTP
GDP
E
5
Start codon
mRNA binding site
3
Small
ribosomal
subunit
A
5
Translation initiation complex
3
• once the small subunit is positioned - the large subunit then
assembles and completes the ribosomal “machine”
• helped by even more eIF’s
• the mRNA and the ribsosome form the Translation Initiation
Complex
• the eIF’s are released once this complex forms
• the ribosome is now ready for the next AA - elongation
follows
Large
ribosomal
subunit
3 U A C 5
5 A U G 3
P site
P i

Initiator
tRNA
mRNA
GTP
GDP
E
5
Start codon
mRNA binding site
3
Small
ribosomal
subunit
A
5
Translation initiation complex
3
2. Elongation
of Translation
http://www.youtube.com/watc
h?v=5bLEDd-PSTQ
http://www.youtube.com/watc
h?v=Ikq9AcBcohA
http://www.youtube.com/watc
h?v=NJxobgkPEAo
2. Elongation
of Translation
3. Termination of Translation
Release
factor
Free
polypeptide
5
3
5
2
3
GTP
2
Stop codon
(UAG, UAA, or UGA)
5
GDP  2 P
-translation also stops at specific codons = STOP codons
-UAA, UGA, UAG
-so when the ribosome reaches these sequences – no more AAs are added and the
ribosome detaches from the peptide strand and mRNA
-a release factor cleaves the polypeptide chain from the tRNA and releases it from the
ribosome (GTP hydrolysis)
-the translation machine “breaks apart” – requires an enzyme that uses ATP hydrolysis
3
•
•
Polyribosomes
a number of ribosomes can
translate a single mRNA
simultaneously, forming a
polyribosome (or polysome)
polyribosomes enable a cell to
make many copies of a
polypeptide very quickly
Completed
polypeptide
Growing
polypeptides
Incoming
ribosomal
subunits
Start of
mRNA
(5 end)
End of
mRNA
(3 end)
(a)
Ribosomes
mRNA
(b)
0.1 m