Transcript Translasyon

Protein Synthesis
Translating the Message
• How does the sequence of mRNA translate into the
sequence of a protein?
• What is the genetic code?
• How do you translate the "four-letter code" of
mRNA into the "20-letter code" of proteins?
• And what are the mechanics like? There is no
obvious chemical affinity between the purine and
pyrimidine bases and the amino acids that make
protein.
• As a "way out" of this dilemma, Crick proposed
"adapter molecules" - they are tRNAs!
The Collinearity of Gene
and Protein Structures
• Watson and Crick's structure for DNA,
together with Sanger's demonstration that
protein sequences were unique and specific,
made it seem likely that DNA sequence
specified protein sequence
• Yanofsky provided better evidence in 1964: he
showed that the relative distances between
mutations in DNA were proportional to the
distances between amino acid substitutions in
E. coli tryptophan synthase
Elucidating the Genetic Code
• How does DNA code for 20 different
amino acids?
• 2 letter code would allow for only 16
possible combinations.
• 4 letter code would allow for 256
possible combinations.
• 3 letter code would allow for 64
different combinations
• Is the code overlapping?
• Is the code punctuated?
The Nature of the Genetic Code
• A group of three bases codes for
one amino acid
• The code is not overlapping
• The base sequence is read from a
fixed starting point, with no
punctuation
• The code is degenerate (in most
cases, each amino acid can be
designated by any of several
triplets)
How the code was broken
• Assignment of "codons" to their respective
amino acids was achieved by in vitro
biochemistry
• Marshall Nirenberg and Heinrich Matthaei
showed that poly-U produced
polyphenylalanine in a cell-free solution from
E. coli
• Poly-A gave polylysine
• Poly-C gave polyproline
• Poly-G gave polyglycine
• But what of others?
Getting at the Rest of the Code
• Work with nucleotide copolymers (poly (A,C),
etc.), revealed some of the codes
• But Marshall Nirenberg and Philip Leder
cracked the entire code in 1964
• They showed that trinucleotides bound to
ribosomes could direct the binding of specific
aminoacyl-tRNAs
• By using C-14 labelled amino acids with all
the possible trinucleotide codes, they
elucidated all 64 correspondences in the code
Features of the Genetic Code
• All the codons have meaning: 61 specify amino
acids, and the other 3 are "nonsense" or "stop"
codons
• The code is unambiguous - only one amino acid is
indicated by each of the 61 codons
• The code is degenerate - except for Trp and Met,
each amino acid is coded by two or more codons
• First 2 codons of triplet are often enough to specify
amino acid. Third position differs
• Codons representing the same or similar amino acids
are similar in sequence (Glu and Asp)
tRNAs
• tRNAs are interpreters of
the genetic code
• Length = 73 – 95 bases
• Have extensive 2o
structure
• Acceptor arm – position
where amino acid
attached
• Anticodon –
complementary to mRNA
• Several covalently
modified bases
• Gray bases are conserved
between tRNAs
tRNAs: 2o vs 3o Structure
Third-Base Degeneracy
• Codon-anticodon pairing is the crucial
feature of the "reading of the code"
• But what accounts for "degeneracy": are
there 61 different anticodons, or can you
get by with fewer than 61, due to lack of
specificity at the third position?
• Crick's Wobble Hypothesis argues for the
second possibility - the first base of the
anticodon (which matches the 3rd base of
the codon) is referred to as the "wobble
position"
The Wobble Hypothesis
• The first two bases of the codon make normal
H-bond pairs with the 2nd and 3rd bases of
the anticodon
• At the remaining position, less stringent rules
apply and non-canonical pairing may occur
• The rules: first base U can recognize A or G,
first base G can recognize U or C, and first
base I can recognize U, C or A (I comes from
deamination of A)
• Advantage of wobble: dissociation of tRNA
from mRNA is faster and protein synthesis
too
AA Activation for Prot. Synth.
• Codons are recognized by aminoacyl-tRNAs
• Base pairing must allow the tRNA to bring its
particular amino acid to the ribosome
• But aminoacyl-tRNAs do something else: activate
the amino acid for transfer to peptide
• Aminoacyl-tRNA synthetases do the critical job
- linking the right amino acid with "cognate"
tRNA
• Two levels of specificity - one in forming the
aminoacyl adenylate and one in linking to tRNA
Aminoacyl-tRNA Synthetase
Amino acid + tRNA + ATP  aminoacyl-tRNA + AMP + PPi
•
Most species have at least 20 different aminoacyltRNA synthetases.
•
Typically one enzyme is able to recognize multiple
anticodons coding for a single amino acids (I.e serine 6
different anticodons and only one synthetase)
•
Two step process:
1) Activation of amino acid to aminoacyladenylate
2) Formation of amino-acyl-tRNA
Aminoacyladenylate Formation
NH2
N
N
N
N
O
O
O
H
H OH
H
P
O
O
O-
P
O-
O
O
P
O-
ONH2
H
OH
O
N
N
O
C
CH
N
H
N
O
PPi
NH2
O
O
H
H OH
H
P
O-
O
H
OH
O
C
CH
NH2
H
Aminoacyl-tRNA Synthetase Rxn
NH2
NH2
N
N
N
N
5' tRNA
N
N
O
O
O
H
H OH
H
H
OH
P
O-
O
H
C
N
O
O
O
N
H
H
O
H
OH
H
CH H
NH2
NH3+
N
AMP
5' tRNA
N
O
O
H
O
H
H
O
H
OH
C
CH H
NH3+
N
N
Specificity of AminoacyltRNA Synthetases
• Anticodon and structure features of
acceptor arm of specific tRNAs are
important in enzyme recognition
• Synthetases are highly specific for
substrates, but Ile-tRNA synthetase has
1% error rate. Sometimes incorporates Val.
• Ile-tRNA has proof reading function. Has
deacylase activity that "edits" and
hydrolyzes misacylated aminoacyl-tRNAs
Translation
• Slow rate of synthesis (18 amino acids per
second)
• In bacteria translation and transcription are
coupled. As soon as 5’ end of mRNA is
synthesized translation begins.
• Situation in eukaryotes differs since
transcription and translation occur in different
cellular compartments.
Ribosomes
• Protein biosynthetic machinery
• Made of 2 subunits (bacterial
30S and 50S, Eukaryotes 40S
and 60S)
• Intact ribosome referred to as
70S ribosome in Prokaryotes
and 80S ribosome in
Eukaryotes
• In bacteria, 20,000 ribosomes
per cell, 20% of cell's mass.
• Mass of ribosomes is roughly
2/3 RNA
Prokaryotic Ribosome Structure
• E. coli ribosome is 25 nm
diameter, 2520 kD in mass, and
consists of two unequal subunits
that dissociate at < 1mM Mg2+
• 30S subunit is 930 kD with 21
proteins and a 16S rRNA
• 50S subunit is 1590 kD with 31
proteins and two rRNAs: 23S
rRNA and 5S rRNA
Eukaryotic Ribosome Structure
• Mitochondrial and chloroplast
ribosomes are quite similar to
prokaryotic ribosomes, reflecting
their supposed prokaryotic origin
• Cytoplasmic ribosomes are larger
and more complex, but many of the
structural and functional properties
are similar
• 40S subunit contains 30 proteins and
18S RNA.
• 60S subunit contains 40 proteins and
3 rRNAs.
Ribosome Assembly
• Assembly is coupled w/ transcription and prerRNA processing
Ribosome Structure
• Crystal structure of ribosome
is known
• mRNA is associated with the
30S subunit
• Two tRNA binding sites (P and
A sites) are located in the cavity
formed by the association of
the 2 subunits.
• The growing peptide chain
threads through a “tunnel”
that passes through the 40S
(30S in bacteria) subunit.
Mechanics of Protein Synthesis
• All protein synthesis involves three phases:
initiation, elongation, termination
• Initiation involves binding of mRNA and initiator
aminoacyl-tRNA to small subunit, followed by
binding of large subunit
• Elongation: synthesis of all peptide bonds - with
tRNAs bound to acceptor (A) and peptidyl (P)
sites.
• Termination occurs when "stop codon" reached
Identification of Initiator Codon in
Prokaryotes
• Involves binding of initiator tRNA (Nformylmethionyl-tRNA) to initiator codon (first AUG)
• The 30S subunit scans the mRNA for a specific
sequence (Shine-Dalgarno Sequence) which is just
upstream of the initiator codon. 16S RNA is involved in
recognition of S-D sequence.
Prokaryotic Translational Initiation
• Formation of Initiation
complex involves protein
initiation factors
• IF-3 keeps ribosome
subunits apart
• IF-2 identifies and binds
initiator tRNA. IF-2 must
bind GTP to bind tRNA.
• IF-1, IF-2, and IF-3 bind to
30S subunit to form
initiation complex
• Once 50S subunit binds
initiation complex, GTP is
hydrolyzed, initiator tRNA
enters P-site and IFs
disassociate
Eukaryotic Initiation of
Translation
• No S-D sequence.
• CAP binding protein (CBP) 5’ end of
mRNA by binding to 5’ CAP structure
• An initiation complex forms with CBP,
initiation factors and the 40S subunit.
• The complex then scans the mRNA
looking for the first AUG closest to the 5’
end of the mRNA
• eIF-2 analogous to IF-2, transfers tRNA
to P sight. GTP hydrolysis involed in
release
Chain Elongation
Three step process:
1) Position correct aminoacyl-tRNA at acceptor
site
2) Formation of peptide bond between
peptidyl-tRNA at P site with aminoacyltRNA at A site.
3) Shifting mRNA by one codon relative to
ribosome.
• Elongation Factor Tu
(EF-Tu) binds to
aminoacyl-tRNA and
delivers it to the A site of
the ribosome
• When EF-Tu binds GTP
a conformational change
occurs allowing it to
bind to aminoacyl-tRNA.
• EF-Tu-tRNA complex
enters the ribosome and
positions new tRNA at
A site.
• If the anticodon
matches the codon,
GTP is hydrolyzed and
EF-Tu releases the
tRNA and then exits the
ribosome.
Recycling of EF-Tu
• After leaving the
ribosome EF-Tu-GDP
complex associates
with EF-Tscausing
GDP to disassociate.
• When GTP bind to the
EF-Tu/EF-Ts complex,
EF-Ts disassociates
and EF-Tu can bind
another tRNA
Peptide Bond formation
P-Site
N
5' tRNA
N
O
H
H+
O
O
C
N
5' tRNA
H
OH
H
O
CH H
NH3+
N
N
N
O
H
5' tRNA
H
H
O
H
OH
N
N
H
H
OH
H
OH
5' tRNA
N
O
H
C
N
O
H
H
O
H
OH
C
CH H
H
BASE
NH2
N
N
O
O
H
A-Site
NH2
N
O
CH H
H
P-Site
NH2
N
N
O
O
H
A-Site
NH2
H
N
O
C
H
H
CH H
NH3+
N
N
Formation of
Peptide Bond
• Once the peptide bond forms,
the mRNA band shifts to
move the new peptidyl-tRNA
into the P-site and moves the
deaminacyl-tRNA from the
E-site
• Binding of EF-GTP to
ribosome promotes the
translocation
• Hydrolysis of EF-GTP to EFGDP is required to release EF
from ribosome and new cycle
of elongation could occur
More on elongation
• Growing peptide chain then
extends into the “tunnel” of the
50S subunit.
• Floding of the native protein does
not occur until the peptide exits
the “tunnel”
• Folding is facilitated by
chaperones that are associated
with the ribosome
• To ensure the correct tRNA enters
the A site, the 16S RNA is involved
in determing correct
codon/anticodon pairing at
positions 1 and 2 of the codon.
Eukaryotic elongation process
•
•
•
•
•
Similar to what occurs in prokaryotes.
Analogous elongation factors.
EF-1a = EF-Tu  docks tRNA in A-site
EF-1b = EF-Ts  recycles EF-Tu
EF-2 = EF-G  involved in translocation
process
Peptide Chain Termination
• Proteins known as "release factors" recognize the stop
codon (UGA, UAG, or UAA) at the A site
• In E. coli RF-1 recognizes UAA and UAG, RF-2
recognizes UAA and UGA.
• RF-3 binds GTP and enhances activities of RF-1 and –
2.
• Presence of release factors with a nonsense codon at A
site transforms the peptidyl transferase into a
hydrolase, which cleaves the peptidyl chain from the
tRNA carrier
• Hydrolysis of GTP is required for disassociation of RFs,
ribosome subunit and new peptide
Protein Synthesis is Expensive!
• For each amino acid added to a
polypeptide chain, 1 ATP and 3 GTPs are
hydrolyzed.
• This is the release of more energy than is
needed to form a peptide bond.
• Most of the energy is need to over-come
entropy losses
Regulation of Gene Expression
RNA Processing
5’CAP
Active
enzyme
Post-translational
modification
mRNA
AAAAAA
RNA Degradation
Protein Degradation
Regulation of Protein Synthesis
Regulation could occur at two levels in
translation
1) Initiation – formation of the initiation
complex
2) Elongation – elongation could be stalled
by if an mRNA contains “rare” codons
Regulation of Globin
gene translation by
heme
• When heme is low, HCI
kinase phosphorylates
eIF-2-GDP complex,
• GEF binds tightly to
phosphorylated eiF-2GDP complex
• prevents recycling of eIF2-GDP and stops
translation
Regulation of the trp operon
• Transcription and translation are tightly
coupled in E. coli.
• When Trp is aundant, transcription of the
trp operon is repressed.
• The mechanism of this repression is related
to translation of the