Chapter 20 Protein synthesis

1
Chapter 20 - Protein synthesis
Central dogma of molecular biology
3 base pairs DNA gt 3 bases of mRNA gt 1
amino acid
2
Translation refers to the process of protein
synthesis, where a ribosome reads a messenger RNA
(mRNA) and synthesizes a protein.
3
Stages of translation
Initiation - mRNA binds to the small subunit of
ribosome, and then the large subunit
joins. Elongation - The protein is synthesized
by the ribosome, using amino acids delivered by
tRNA. Termination - The newly synthesized
protein is released from the ribosome.
4
Genetic code
DNA structure was known by 1953, but how it coded
for proteins was unknown. Artificial mRNA
templates were made and used to break the code.
For example, it was observed in 1961 that poly
U made poly-phenylalanine. poly U gt poly
Phenylalanine poly G gt poly Glycine
Poly A gt poly Lysine poly C gt poly Proline
5
Some amino acids have one codon, some have 3, 4,
or 6 codons.
Interestingly, a change in the 3rd nucleotide
often does not change the amino acid, or changes
to a similar a.a.
6
Reading frames.
Notice there are 3 different reading frames for
the triplet code. The a.a. translated depends on
where the ribosome starts.
Can you see how a mutation that deletes a single
nucleotide could change the identities of all the
amino acids in a protein?
7
Amino acids are delivered to the ribosome
attached to the 3 end of transfer RNA (tRNA) at
the acceptor stem. The anticodon loop of tRNA
reads the mRNA.
All tRNAs have a similar cloverleaf
structure. All end in -CCA at their 3 end
(where the amino acid is attached).
8
tRNAs contain some modified nucleotides
(dihydrouridine, etc.)
9
Steps in
translation Amino acid activation - attaching
the a.a. to the 3 end of tRNA. Initiation -
find the start codon, and start
translation. Elongation - add a.a. to the
growing peptide chain. Termination - At the
stop codon, release the ribosome from mRNA.
10
Enzymes called aminoacyl tRNA synthetases link
the a.a. to the 3 end of the tRNA. A different
tRNA synthetase for each tRNA type, to assure the
correct a.a. is coupled to the correct
tRNA. Amino acid attachment requires ATP.
Gln tRNA synthetase (green) bound to tRNA (red)
and ATP (yellow)
FYI - there are 2 classes of tRNA synthetases
(called class 1 and class 2) which operate
by 2 distinct mechanisms.
11
tRNA with a.a. attached
12
activated amino acid
charged tRNA
13
Proofreading by the a.a. tRNA synthetase keeps
error rates low. For example, about 1/50,000
reactions results in an Isoleucine tRNA being
aminoacylated with valine.
14
The anticodon loop of the tRNA uses base pairing
to read the genetic code on the messenger RNA.
15
The 3rd nucleotide in each codon is called the
wobble base. Bacteria use typically 40 tRNAs
to read the 64 possible codons. How do they do
that? With wobble base pairing, so one tRNA
can recognize more than one codon.
16
Wobble base-pairing Some tRNAs can pair with
more than one codon (so 40 tRNAs can read 61
possible codons). The 3rd position wobbles
U base pairs with A or G. G base
pairs with C or U. I (inosine) base pairs
with A, C or U
17
Wobble base-pairing
note I inosine
From wobble base pair wikipedia.
18
(No Transcript)
19
You can think of the ribosome as a big enzyme
that synthesizes protein with a.a. sequence
directed by the mRNA.
ribosome
mRNA gt protein
20
Ribosomes have 2 subunits (small and large).
Small subunit binds mRNA. Linking of the 2 amino
acids (the peptidyl transferase reaction) occurs
on the surface of the large subunit.
21
Naming of ribosome parts.
30S
50S
30S
Bacterial ribosome - Small subunit is called
the 30S subunit. Large subunit is called the
50S. When both subunits are assembled
together, they are called the 70S
ribosome. The small subunit contains the 16S
RNA (1600 nucleotides, called the 16S RNA) and 21
proteins (named S1, S2, S3, etc.) The large
subunit contains the 23S RNA (about 2900
nucleotides) and 5S RNA, and about 31 proteins
(named L1, L2, L3, etc.).
22
With about 52 proteins and 3 RNAs, the bacterial
ribosome has a molecular weight of over 2 million
atomic mass units. The eukaryotic ribosome is
similar in structure, but even larger.
23
Structure of the prokaryotic ribosome is now
known (from x-ray crystallography).
RNA of large subunit is gray, small subunit RNA
is cyan, proteins are purple and blue. tRNAs
held between the 2 subunits are red and yellow.
Yusupov, Yusupova, Baucom, Lieberman, Earnest,
Cate, Noller (2001) Science, 292, 883-896.
24
Where does the ribosome start
translation? Not on the first nucleotide of the
mRNA ! In bacteria, translation starts (almost
always) on the first AUG codon that follows the
Shine-Dalgarno sequence on the mRNA.
Shine-Dalgarno sequence
5 end
Start codon
mRNA
16S RNA of ribosome
25
Initiation of translation (in bacteria).
In bacteria, the first a.a. brought to the
ribosome is N-formylmethionine. It is
delivered by a special tRNA that recognizes the
start codon. Regular a.a. are used after that.
26
Synthesizing the protein - 3 tRNAs are
bound between the 2 ribosome subunits.
A-site and P-site tRNAs are reading mRNA with
their anti-codon loops, and have their bound
amino acids linked together at the peptidyl
transferase center (PTC) of the ribosome. E-site
(exit site) tRNA has donated its amino acid and
is leaving.
27

• The function of the ribosome is to
• hold the tRNAs in position so that the anticodon
  loops can read the mRNA.
• hold the 3 ends of the tRNAs (with bound a.a.)
  in position so the a.a. can be linked in the
  growing peptide chain.

28
At the peptidyl transferase center, amino acids
are added to the growing peptide chain one at a
time.
29
Peptidyl transferase reaction (the linking of
a.a. in the growing peptide chain). The
activated amino acid on the tRNA in the A-site is
added to the growing peptide chain peptide is
transferred to the tRNA in the A-site.
30
Translocation step This is the movement of mRNA
through ribosome to the next codon, pushes tRNA
from the E-site. Empty tRNA in P-site is pushed
to E-site tRNA with growing peptide chain moves
from A-site to P-site next tRNA enters A-site
hydrolysis of GTP on EFG (elongation factor G)
provides energy to help drive translocation.
Protein synthesis requires a lot of energy 2
molecules of GTP for each amino acid added, plus
one ATP for aminoacylating the tRNA.
31
The ribosomal elongation cycle
Figure 20.18.
32
Elongation (in bacteria).
EF-Tu
tRNA
33
Proof reading - Is the right amino acid being
incorporated?
tRNA binding in A site triggers conformational
change in ribosome. 2 nucleotides of 16S rRNA on
small subunit flip out and H-bond with ribose on
mRNA if it is correctly paired with the tRNA. If
not correctly paired, the tRNA is rejected. If
it is correctly paired, this sends a signal
(through conformational change) to hydrolyze GTP
on elongation factor EF-Tu. This GTP hydrolysis
is required for peptide bond to form. The time
lag between tRNA binding and peptide bond
formation is kinetic proofreading to assure
correct a.a. is added.
34
Termination. A
termination codon in the mRNA signals the
ribosome to end translation and release the
mRNA. Release factor proteins recognize the
termination codon, and mimic the tRNA structure,
bind to the ribosome, and trigger its release
from the mRNA.
35
As soon as one ribosome is clear of the start
codon, another ribosome may begin translation.
Polysomes are many ribosomes all translating the
same mRNA at the same time.
36
Some antibiotics bind specifically to bacterial
ribosomes, exploiting small differences between
bacterial and eukaryotic ribosomes (so the
antibiotic kills bacteria without harming us).
Puromycin binds to A-site of bacterial mRNA, and
competes with tRNA attempting to occupy A-site.
37
Chloramphenicol selectively binds bacterial
ribosomes.
38
(No Transcript)
39
(No Transcript)
40
Translation in eukaryotes
Some important differences from bacteria
- Eukaryotic mRNAs are capped, and have poly A
tail. No Shine-Dalgarno sequence, translation
starts on first AUG. Many more initiation factor
proteins. Modification (phosphorylation) of
certain initiation factor proteins is a method of
regulating protein synthesis. On eukaryotic
mRNAs, the 5 end and 3 end are actually close
together when the mRNA is being translated, bound
by initiation factor proteins and poly-A binding
proteins.
41
Folding of newly synthesized proteins is assisted
by chaparone proteins.
42
Proteins are eventually degraded in the
proteasome. In eukaryotes, proteins may be
tagged for fast degradation by having a small
protein called ubiquitin attached.
43
ribosome
mRNA gt protein
Thats the end of the book !!!