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 aminoacyl-tRNA 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 aminoacyladenylate2) Formation of amino-acyl-tRNA

Aminoacyladenylate Formation

O

N

NN

N

NH2

O

OH OH

H H

HH

O P

O-

O

OP

O-

O

O-P

O-

O

NH2

CH

C

H

O

O

PPiO-

N

NN

N

NH2

O

OH OH

H H

HH

O P

O

O

NH2

CH

C

H

O

Aminoacyl-tRNA Synthetase Rxn

N

NN

N

NH2

O

OHO

HH

HH

O

5' tRNA

H

N

NN

N

NH2

O

OHO

HH

HH

O

5' tRNA

NH3+

CH

C

H

O

O-

N

N N

N

NH2

O

OH OH

H H

H H

O P

O

O

NH3+

CH

C

H

O

AMP

Specificity of Aminoacyl-tRNA

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 pre-rRNA 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 (N-formylmethionyl-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 aminoacyl-tRNA 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

N

NN

N

NH2

O

OHO

HH

HH

O

5' tRNA

N

CH

C

H

O

N

NN

N

NH2

O

OHO

HH

HH

O

5' tRNA

NH3+

CH

C

H

O

H

H

H

BASE

H+

P-Site A-Site

N

NN

N

NH2

O

OHO

HH

HH

O

5' tRNA

N

CH

C

H

O

N

NN

N

NH2

O

OHOH

HH

HH

O

5' tRNA

NH3+

CH

C

H

O

HH

P-Site A-Site

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 EF-GDP 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

AAAAAA5’CAPmRNA

RNA Processing

RNA Degradation

Protein DegradationPost-translational modification

Activeenzyme

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-2-GDP complex

• prevents recycling of eIF-2-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