L11 Biol 261 Ftranslation 2014

L11 Translation
Lecture 11
TRANSLATION
Protein synthesis
- Around 1900, Archibald Garrod hypothesized there is a

relationship between inheritance of a visible trait (black urinealkaptonuria) and inheritance of a defective enzyme.

-In the early 1940s Beadle and Tatum concluded that:

one gene governed the expression of one enzyme.

Currently: (1) genes encode more than proteins, e.g. RNA(many
kinds)

(2) one pre-mRNA can be multiply spliced into several
different proteins.

(3) there different kinds of proteins, as well as
enzymes there are structural, signaling and transporting
proteins..

In eukaryotic organisms, one (processed) mRNA typically

encodes a single protein.
Eukaryote
primary
transcript
has to be
modified
and
exported

Bacteria
translation
starts
before the
transcript
is finisned

Bacteria have poly-cistronic mRNAs in which

adjacent genes are transcribed in one message
gene 1
gene 2
gene 3
Reading a gene - Codon and Code

A pre-mRNA is transcribed and processed into a mRNA base

sequence.
cap5CAAGUGAUGACGUAUUCUCGCUAG3tail
20 amino acids: 1 base- 4 possibilities (ATCG),
2 bases 16,
3 bases 64,
(4 * 4 * 4)

An mRNA is translated
in continuous base triplets
called codons.
6
THE GENETIC CODE see

20 amino acids, 61 possibilities ( + 3 stop codons)
Degenerate, synonyomous
The genetic code is (almost)

universal.
Genes can be cloned from plants
and animals and expressed in E.
coli and vice versa.
but
The coding part of a eukaryotic
gene can be accurately
translated in bacteria, but
(1)the promoter sequence has to
be changed to a bacterial
promoter,
AUG start

(2) the introns have to be

removed.
A pre-mRNA is transcribed and processed into a mRNA base

sequence.
cap5CAAGUGAUGACGUAUUCUCGCUAG3tail
20 amino acids: 1 base- 4 possibilities (ATCG), 2-16, 3-64
mRNA is translated using continuous base triplets
called codons.
(1)There is a start signal (AUG).
(2)The codons do not overlap.
(3) There is no space or extra nucleotides between codons in
prokaryote and eukaryotic mRNA.
There are 3 possible stop signals (UAG, UAA, or UGA) .
(4) Direction read mRNA from the start (5 end)

(5) With a triplet genetic code, there are 3 possible ways of

translating, depending on the starting point *.

5
*

3

3

*
5

There are two strands, thus there are 6 possible

reading frames (2*3), but the simplest expectation is

1 gene per duplex sequence.

(6) Identify start within one strand (5-3)

10
(3) With a triplet genetic code, there are 3 possible ways of
translating (5- 3), depending on the starting point (reading frame),
on 2 strands.
First find the start codon (in mRNA
In the non-template or coding strand
In the template strand
5 AUG 3)
5 ATG 3
3 TAC 5
Then find the stop codon (in mRNA

5UAA, UAG, UGA 3)

5 TAA, TAG, TGA 3
3 ATT, ATC, ACT 5
Then determine each amino acid after the start codon and before the
stop codon working with mRNA or the coding strand
First find the start codon in mRNA

5AUG 3
5 ATG 3
3 TAC 5
Then find the stop codon in mRNA

5UAA, UAG, UGA 3

5 TAA, TAG, TGA 3
3 ATT, ATC ACT 5
Template and coding strand ? Gene ?

1

5 GAT CCC ATG GCT ATG TAG CGC

CTA GGG TAC CGA TAC ATC GCG 5

5 GCC TTA GTC CCT AGG CAT AAC

CGG AAT CAG GGA TCC GTA TTG 5

11

Open reading frames (ORF) are located between start and stop
codons or, a nucleotide sequence that does not contain any stop codons.
By chance, a termination codon should occur 3/64 codons, but,
genes produces mRNAs that typically have long open reading frames or, stop
codons occur much less frequently than you would expect by chance.

Template DNA
Open Reading Frame
ORF
TAC
3UTR
3 untranslated region
(leader)
ATT (ATC, ACT)

5UTR
5 untranslated region
(trailer)
12
In a computer search for gene sequence

the base sequence of a DNA molecule is scanned
13
For:
(1) Promoter sequence
(2) Intron exon junctions - GU INTRON AG rule
(3) GC rich islands exons tend to have a higher % G
and C than intergenic regions
(4) Start (ATG) and downstream STOP codons
(5) Long open reading frames (ORFs)
(6) Similarity to related genes known in other species
Confirm a DNA sequence encodes a gene by showing it is transcribed
into mRNA
Bioinformatic scans are a useful tool for identifying potential genes,
but it is often difficult to predict the start codon and intron/exon
boundaries.
A mature mRNA has:
14
Reading Frame
AUG
5UTR
Cap 5 untranslated region
(leader)
UAA, (UAG, UGA)

3UTR
3 untranslated region (trailer)
and a poly A tail
Most mRNAs are between 1000 and 2000 nucleotides (nt)

long though the range of sizes is quite large and can be as
high as 10,000 nt or as low as 100nt.
The translation system consists

of five major components:
!Messenger RNA: mRNA is needed to
provide the coding sequence of bases
that determines the amino acid
sequence in the resulting polypeptide
chain
!Transfer RNA: tRNA is a small
adaptor molecule that mediates
codon translation to amino acids
!Aminoacyl-tRNA synthetases:
molecules that catalyze the
attachment of a particular amino acid
to its corresponding tRNA molecule
!Ribosomes are multi-unit molecules
on which protein synthesis takes place
!Initiation, elongation termination
factors
15

16

tRNA - the adaptors
t RNAs make the

physical link
between the mRNA
codon and the
specific amino acid
corresponding to
each codon.
The specific amino acid
is added to its specific
tRNA by
amino acyl tRNA
synthetase.
3OH
17
18
(3)- release the charged tRNA

(1)

(2)

Where does the initiatorn formyl methionine (or

methionine-tRNA) bind in
the 70s (or 80s ribosome ?)

a) E site

b) P site

c) A site

d) Any of the above

Initiation

Requires:

a) mRNA

binding to a ribosome assembly of:

b) small ribosomal subunit

c) large ribosomal subunit

and

d) initiator tRNA

e) initiation factors coordinating a, b and c

19

There are conserved sequences in the leader region before the initial
AUG in mRNA (5 untranslated region, 5 UTR).
In bacteria and bacteria phage a region critical for initiation is the
Shine-Dalgarno sequence (consensus AGGAGU)
found 5-10 (~10) nucleotides upstream of the initial AUG.
20
The start codon AUG encodes methionine,

21
In bacteria, the first amino acid is modified by the addition of a
formyl group producing N-formylmethionine which is removed
later
Formyl

group

Prokaryote Translation is initiated by

sequential binding to the
mRNA by Initiation Factors
In E. coli, IF-1 and IF-3 initiation factors
recruit the 30S subunit

IF-2 binds with a special tRNA charged with
formyl methionine tRNAfMeti (initiator)

The 30 S initiation complex at the Shine

Dalgarno sequence, (30s subunit, IF1, IF2fmet positioned at AUG), IF3 is released.

The 30s initiation complex recruits a 50S subunit, in the process, IF1, IF2 are released

22
In eukaryotes, translation initiation, the 5

cap on the mRNA is instrumental for binding
(poly A tail enhances initiation) and initiation
takes place by scanning the mRNA for an
initiation codon

23
The initiation factor eIF4 binds to the cap

and recruits other initiation factors. This
creates a binding site for a charged tRNAMeti
(an initiator tRNA), bound with initiation
factor (eIF2,) and a small 40S ribosomal
subunit together with other eIF. These
components all come together at the 5 cap
and form the 40S initiation complex

The initiation complex moves along the

mRNA in the 5- 3 direction, scanning for
the the initial methionine codon AUG

AUG found: At this point eIF5 causes the
release of all the initiation factors before the
recruitment of a large 60S ribosomal subunit
to make the 80s initiation complex-
Degenerate, synonyomous
5 ATGTTTGAATGG 3

3 TACAAACTTACC 5

If the sequence above were

transcribed and translated from

left to right starting at the first
nucleotide, the translated
polypeptide would consist of the
sequence:

a) Tyr-Lys-Leu-Thr.

b) Met-Phe-Glu-Trp.

c) Met-Lys-Glu-stop.

d) Met-Lys-stop.

e) Gly-Lys-Phe-Val.

AUG start

The code is degenerate with more codons

than amino acids, thus there are synonymous
substitutions. How many TRNAs does this
suggest there are?

a) 61

b) > 61

c) 20- 40

d) 20

E < 20

Degeneracy (1) number of codons vary from 1 to as

many as 6, most amino acids have alternative tRNA
types (anticodon-codon pairing, synonomous because they
produce the same protein).

But.
(2) Are there tRNAs for every codon?

No! 30 ~ 40 in E. coli

Wobble in third position (3-5) anticodon or one
anticodon can recognize several codons

I = inosine use it in plant tRNA.

serine

But not all theoretical

wobble pairing exist,

just those which
account for the code
universality have been
observed.

Johansson et al 2012 PNAS 109

Accuracy and efficiency tradeoff

Second position is most tightly controlled and controls

chemical properties of incoming amino acid, 3rd position
wobble,

Justifies:

(1) Proof reading by the ribosome

Explains in part:
(2) Codon bias (species have greater
frequency of one codon than expected by chance) and tRNA
optimization (higher expression of 1 form)

Elongation:

24
3 steps, 3 sites, 2 elongation factors, 1 enzyme

(1) Aminoacyl-tRNA(charged) binds to
the ribosome in the A site (+elongation factor
TuGTP)

(2) A peptide bond forms with the
amino acid attached to the tRNA in the P site.
(Pepitdyltransferase is the 23S rRNA of the large
subunit).

(3) elongation factor GGTP inserts at A,
Gtp is de-phosphorolated, the ribosome large
subunit rocks along mRNA by 1 codon, the
tRNA in the E site exits

(4) the small subunit snaps back into
position with the dephosphorolation of GTP

Making
proteins
costs a lot
of energy

Translocation

25

(1)

EF-TuGTP

+TRNA

Peptidyltransferase- 23sRNA

(2)

EF-GGTP

(3)

(4)

26

27
Eukaryotic Elongation
eukaryotic analogues
EF-tu
is
EF-Ts (recharges EF-tu) is
eEF-1
eEF1
EF-G
eEF-2
is
TERMINATION

Termination
When a stop codon is encountered, the
tRNA holding the polypeptide remains in
the P site, and a protein release factor (RF)
binds with the ribosome.

There are three release factors in E. coli

One for each stop codon

Eukaryotes have only one release factor
that recognizes all three stop codons:
UAA, UAG, and UGA

GTP hydrolysis provides the energy to
cleave the polypeptide from the tRNA to
which it is attached

The 40S and 60S subunits are recycled
to initiate translation of another mRNA

28
29

Polysome
(polyribosome)

mRNA is translated in the 5-to-3 direction. A polypeptide is

synthesized from the amino end toward the carboxyl end
30
31
32

Figure 3-23 Molecular Biology of the Cell ( Garland Science 2008)

Levinthals paradox.

Cyrus Levinthal (1969) noted that because there are several
conformations possible with each peptide bond in an unfolded
polypeptide chain, even a small polypeptide sampling the
number of possible conformations could take longer than the
estimated age of the universe to reach its native state.

But, most small proteins fold spontaneously on a millisecond or even
microsecond time scale.

Consider a protein emerging from a ribosome, it is in an aqueous,
slightly salty (charged) environment crowded with other proteins,
lipids, mRNA and so on, all of which might interact with the
incompletely-formed protein.

Part of the solution to this paradox is there are folding catalysts
and molecular chaperones that assist in the process of folding to
its native state.

Few polypeptide chains fold correctly as they exit the ribosome: they pass through a
tunnel in the large ribosomal subunit that is long enough to include about 35 amino
acids (self-assembly). Emerging from the tunnel, protein enters into a sort of cradle
formed by a protein associated with the ribosome: it provides a space where a small
polypeptide is able to undergo its inherent folding process.

The proper folding of more complex polypeptides is aided by proteins called
chaperones and chaperonins of 2 kinds: (1) small proteins that cluster around the
exit, (2) a subsequent chamber.
39
What it takes to express 1

protein, a molecular
phenotype.

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L11 Translation

- Around 1900, Archibald Garrod hypothesized there is a

In eukaryotic organisms, one (processed) mRNA typically

Bacteria have poly-cistronic mRNAs in which

Reading a gene - Codon and Code

A pre-mRNA is transcribed and processed into a mRNA base

THE GENETIC CODE see

The genetic code is (almost)

(2) the introns have to be

A pre-mRNA is transcribed and processed into a mRNA base

(4) Direction read mRNA from the start (5 end)

(5) With a triplet genetic code, there are 3 possible ways of

There are two strands, thus there are 6 possible

Then find the stop codon (in mRNA

5UAA, UAG, UGA 3)

First find the start codon in mRNA

Then find the stop codon in mRNA

5UAA, UAG, UGA 3

Template and coding strand ? Gene ?

5 GAT CCC ATG GCT ATG TAG CGC

ATT (ATC, ACT)

In a computer search for gene sequence

A mature mRNA has:

UAA, (UAG, UGA)

Most mRNAs are between 1000 and 2000 nucleotides (nt)

The translation system consists

tRNA - the adaptors

t RNAs make the

(3)- release the charged tRNA

Where does the initiatorn formyl methionine (or

The start codon AUG encodes methionine,

Prokaryote Translation is initiated by

The 30 S initiation complex at the Shine

In eukaryotes, translation initiation, the 5

The initiation factor eIF4 binds to the cap

The initiation complex moves along the

If the sequence above were

The code is degenerate with more codons

Degeneracy (1) number of codons vary from 1 to as

But not all theoretical

Johansson et al 2012 PNAS 109

Second position is most tightly controlled and controls

3 steps, 3 sites, 2 elongation factors, 1 enzyme

There are three release factors in E. coli

mRNA is translated in the 5-to-3 direction. A polypeptide is

What it takes to express 1

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