Lecture 24- Protein translation II

Protein synthesis

-requires the specific binding of mRNAs and aminoacyl-tRNAs to the ribosome, a large multiprotein/RNA complex that is the site of protein synthesis

ribosomes

-contain a large percentage of ribosomal RNA (rRNA)

-it is now widely accepted that the rRNA component is the site of catalytic activity; this makes a ribosome a ribozyme

-in prokaryotes, ribosomes contain 2 separate subunits that are classified by their sedimentation coefficients (50S and 30S)

protein synthesis in all organisms

-starts at the N-terminus and proceeds in the N→C direction

N-terminal amino acid in prokaryotes

-in most proteins is N-formylmethionine (fMet); fMet is typically removed during post-translational processing

-indicates that prokaryotes have 2 tRNAs for methionine: tRNA^Met and tRNA^fMet

To charge tRNA with fMet

-Met first binds to tRNA^fMet, generating Met-tRNA^fMet, and is modified with a formyl group while bound to the tRNA; this reaction is catalyzed by Met-tRNA formyl transferase

tRNA^Met and tRNA^fMet

-both contain 3’-UAC-5’ sequences that recognize 5’-AUG-3’ of the mRNA; typically, this codon serves as the start codon that directs synthesis of the polypeptide chain

beginning of protein synthesis

-begins with the formation of the initiation complex; at least 8 components are involved: mRNA, fMet-tRNA^fMet, GTP, 30S subunit and various initiation factos )IF-1, IF-2, IF-3, …)

-the 30S subunit is initially bound to initiation factors IF-1 and IF-3 and recruits fMet-tRNA^fMet and IF-2, generating the 30S initiation complex

-this complex then binds the mRNA at an upstream Shine-Dalgarno sequence; this sequence is analogous to a promoter on DNA, which directs the ribosome to the initiator start site (AUG)

-once bound to the mRNA, the complex recruits the 50S ribosomal subunit, forming the 70S initiation complex; the formation of this complex involves the hydrolysis of GTP and the concomitant release of IF-1 and IF-3

3 binding sites recognized by tRNA on the 70S ribosome

-A-site

-P-site

-E-site

A-site

-“aminoacyl (A) site”

-binds a new incoming charged tRNA that corresponds to the adjacent codon on the mRNA

P-site

-“Peptidyl (P) site”

-the site of catalysis where a new peptide bond is formed between 2 amino acids

E-site

-“exit (E) site”

-where the uncharged tRNA is ejected from the complex

chain elongation

-begins with the addition of the second amino acid (fMet is the first) in the sequence; each amino acid is designated by the sequence in the mRNA

4 main steps of elongation

1.A new aminoacyl tRNA from the environment binds to the A site of the ribosome; this step requires GTP and an elongation factor (EF-Tu)

2.EF-Tu is released from the ribosome and regenerated using GTP and EF-Ts

3.a new peptide bond is formed by nucleophilic attack of the ɑ-amino group of the A-site aminoacyl-tRNA on the carbonyl of the P-site peptidyl-tRNA

4.translocation occurs when the entire complex shifts over by 1 codon; this causes the uncharged tRNA to enter the E-site (ejected), while the A-site peptidyl-tRNA enters the P-site

termination of peptide synthesis

-requires a specific signal (stop codon)

-codons UAA, UAG, and UGA are all stop codons; these codons are not recognized by any known tRNA

-stop codons are recognized by release factors (RF-1, RF-2, …); once a stop codon is reached in the mRNA sequence, RF-1 or RF-2 is bound by the ribosome at the A-site

-The binding of RF-1 or RF-2 effectively blocks any new aminoacyl-tRNAs from binding to the A-site and facilitates the hydrolysis of the peptidyl-tRNA in the P-site; following hydrolysis of the peptide, the entire complex dissociates

x-ray crystal structure of the ribsosome

-showed that there is no protein in the vicinity of the peptidyl transferase site (P-site)

-this coupled with the discovery of self-splicing RNA indicated that RNA itself can have catalytic activity, thus the ribosome is a ‘ribozyme’

-still questioned because mutations within the ribosome at the catalytic bases do not change its catalytic efficiency

in prokaryotes

-several ribosomes can be attached to a single mRNA strand simultaneously; these structures have been dubbed polysomes

-while each ribosome only produces 1 copy of the protein coded by the mRNA, this efficient mechanism allows multiple copies of a single protein to be produced simultaneously

-the mRNA in polysomes is sometimes directly linked to DNA (being transcribed) and represents an efficient process by which protein can be made from DNA very rapidly

-this process is called coupled translation and is possible in prokaryotes because they do not contain any compartmental separation between DNA, RNA, and proteins