Download Lecture 25: Protein Synthesis

Lecture 25: Protein Synthesis
Key learning goals:
• Be able to explain the main stuctural features of
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ribosomes, and know (roughly) how many DNA and protein
subunits they contain.
Understand the main functions of the big subunit.
Understand the main functions of the lile subunit.
Explain what a polysome is.
Understand how ribosomes place themselves on the start
codon. This is different in bacteria and in eukaryotes. Be
able to compare and contrast these mechanisms.
Understand what bacterial IF-1 and IF-3 do.
Understand what the eukaryotic eIF4 complex does.
Understand what a polycistronic mRNA is. Be able to
explain why polycistronic mRNA’s are very common in
bacteria, and extremely rare in eukaryotes.
Be able to explain why coupled transcription and
translation occur in bacteria, but not in eukaryotes.
Understand the special role of formylated methionine
(fMet) in bacterial initiation.
Template-mediated polymer synthesis: 3 stages
(2%
62%
TVSXIMR
Initiation. Locate the starting
point on the template; assemble
the polymerization machinery.
Elongation. Add a protomer to
the growing polymer, as specified
by the sequence on the template;
repeat many times.
Termination. Cease elongation;
disassemble the elongation
hardware.
TRANSLATION PARTS LIST:
• mRNA — the template
• amino acids (20)
• tRNAs (approximately 40)
• aminoacyl tRNA synthases (20)
• ATP
• GTP
• ribosome
-small subunit (decoding center)
-large subunit (peptidyl transferase center)
• initiation factors
• elongation factors
• termination factors
Ribosomes & Protein Synthesis
This diagram summarizes a lot of what you will need to know about bacterial protein synthesis.
Venki Ramakrishnan
erythrocyte
8,000 nm
bacterium
2,000 nm
300 bp DNA 2 nm wide
100 nm long
ribosome
25 nm
= 10 nm = 100 Å
David Goodsell
prokaryotes
eukaryotes
makes the
polypeptide
makes the
polypeptide
decodes the
mRNA
decodes the
mRNA
Harry Noller
The 70 S bacterial ribosome
Noller Group, UCSC
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http://rna.ucsc.edu/rnacenter/ribosome_rht.html
Ribosomal proteins lie mainly on the surface
The folding of ribosomal subunits is highly conserved
Crystal Structure of the Eukaryotic 40S Ribosomal Subunit in Complex with Initiation Factor 1
Julius Rabl, et al. Science 331, 730 (2011)
Evolutionary conservation & divergence
of ribosomal proteins
Proteins of the small subunit. Cores found in all kingdoms are light blue. Archaeal
cores are orange. Proteins or extensions uniquely found in eukaryotes are red.
Rabl et al., Science 331:730
Distinctive features of the
eukaryotic ribosome map to the
cytoplasmic surface
40 S
Eukaryote-specific RNA
loops in red; eukaryotespecific proteins in yellow.
60 S
Conserved region surrounding
polypeptide exit tunnel
Ben-Shem et al., Science 334:1524
mRNA with multiple translating ribosomes: a polysome
100 nm
mRNA with multiple translating ribosomes: a polysome
3´
5´
100 nm
Three Stages in Translation
Initiation: the ribosome is placed on the start codon
Elongation: mRNA-templated polypeptide polymerization
Termination: the polypeptide and mRNA are released
@
M2.!
AUG
RIBOSOME
POLYPEPTIDE
.(
@
@
@
@
@
$.!
2.! POLYMERASE
Note: this cartoon applies to bacteria. In
eukaryotes, transcription occurs in the
nucleus, translation in the cytoplasm. But
the directions are the same in all cases.
In bacterial initiation, ribosome small subunit binds directly
to Shine-Delgarno initiation sites on the mRNA
In eukaryotic initiation, the small subunit binds the
7-methyl-G cap, then scans 5´ to 3´ to find a start codon
To understand initiation, you first must
understand the basics of elongation!
The ribosome has three
tRNA binding sites:
1. Aminoacyl-tRNA
2. Peptidyl-tRNA
3. Exit
Direction of
tRNA and mRNA
movement through
ribosome
EPA
Three major steps in elongation:
A site:
tRNA selection
P site:
peptidyl transfer
translocation:
uncharged tRNA
exits from E site
Note that the growing
polypeptide chain is
transferred onto the
incoming aa-tRNA!
The aa on the incoming
aa-tRNA is not transferred
onto the chain!
Initiation in bacteria
Ingredients:
mRNA
fMet-tRNA[fMet]
initiation factors
IF-1
IF-2
IF-3
GTP and Mg2+
Small (30 S) subunit
Shine-Delgarno
sequence binds
small subunit
AUG start codon
RNA
aligned in the P site
IF-1 occupies
the small subunit’s
A site
Initiation in bacteria
fMet
Bacteria use a specialized initiator
tRNA charged with a modified amino
acid, N-formylmethionine (fMet).
Eukaryotes use plain old Met.
The presence of peptides containing N-terminal
fMet is interpreted by animal immune systems
as a sign that bacteria are present or that
mitochondria have ruptured.
In other words, for us fMet is a danger signal.
Initiation in bacteria
Starting state for elongation:
• Initiation factors have fallen off
• Large (50S) subunit bound
• fMet-tRNA and AUG codon are in P site
• A and E sites empty
Eukaryotic initiation: the small subunit scans
from 5´cap until it finds a start codon
As a consequence, eukaryotic mRNAs are almost always monocistronic:
they contain only a single initiation site and encode only one polypeptide.
polycistronic
AUG
AUG
AUG
monocistronic
AUG
✁ ✁
In some cases, a polyprotein can be
cleaved by site-specific proteases to
yield more than one polypeptide
Initiation in eukaryotes
Ingredients:
mRNA
Met-tRNA
initiation factors:
eIF2 > know what this does
eIF4 > know what this does
eIF5
eIF6
GTP, ATP, and Mg2+
40 S subunit
eIF4 complex binds mRNA 5´ cap & poly-A tail
Initiation in eukaryotes
not fMet
P A
Small subunit
scans for AUG
ATP consumption!
Initiation in eukaryotes
Starting state for elongation
• eIF4 complex stays bound to 5´ cap
• Other initiation factors have fallen off
• Large (60S) subunit bound
• Met-tRNA and AUG codon are in P site
• A and E sites empty