Translation

Contents

mRNA

mRNA is not a passive player in translation. It is a nucleic acid, and as such contains digital information that can target the mRNA to different parts of the cell, and has a secondary and tertiary structure that can attract the attentions of regulatory proteins.

In eukaryotes, even before mRNA leaves the nucleus, it is extensively modified from the pre-mRNA that was synthesised by RNA polymerase. mRNA is capped at its 5′ end and complexed to cap binding proteins. It is spliced by U-particles, which tag mRNA and the spliced out lariats with different hnRNPs: SR-rich proteins bind exons and hnRNPs bound to excised lariats package them up and mark them for destruction. The 3′ tail of mRNA has a poly-A tail added, which is bound by PABP. All this must occur before export factors binds, and all these items are required for appropriate export from the nucleus.

Export-ready RNA is really a ribonucleoprotein, with many associated proteins and RNPs.

mRNA can be targeted to specific places in the cytoplasm: the targeting sequences are found in the 3′ UTR (untranslated region).

UTRs.
The 5′ UTR contains the ribosome binding site. The 3′ UTR contains mRNA targeting sequences.

Targeting is essential to protein function: nerve cells in the hypothalamus (which secrete hormones from their dendrites) target their mRNAs. Vasopressin mRNA is directed to dendrites by a 400 nucleotide long sequence in the 3′ UTR. It appears that PABP, bound to this region, binds to cytoskeletal motors. Other mRNAs are also targeted: calmodulin dependent protein kinase II has a shorter 'Y' element (below), bound by TB-RBP (testis-brain RNA binding protein).

5′---AUG---UAG---AGAAGCCCTATGCT---AAAA---3′

The cytoplasm contains RNAses, and RNA is in any case inherently unstable to hydrolysis. However, different mRNAs have different stabilities: β-globin mRNA in young erythrocytes is very stable; whereas growth factor mRNA is very short-lived (t½ = 30 min). The length of the poly-A tail regulates half-life: it is slowly trimmed off like a time-fuse by an enzyme known as DAN. Some selected mRNAs have poly-A re-added (especially in oöcytes).

Histone mRNA has a protective stem-loop rather than a poly-A tail.

Histone mRNA is unusual in that it lacks a poly-A tail and has a much shorter half-life (about 1 hr) than most poly-A mRNAs. Rather than having a poly-A tail, histone mRNA has protective stem-loop structure at its 3′ end. Histone mRNA is rapidly degraded at the end of S-phase (its half life drops to just 12 min).

Some mRNAs are also regulated by endonucleases that chop off the tail wholesale. This is seen in the regulation of the mRNAs of iron metabolism:

Iron metabolism is regulated at the level of mRNA stability.

Translation

Before going any further, a few possible misconceptions will be addressed. Translation vs. transcription: Message → Message → સંદેશો. Transcription is 'easy': DNA and RNA are different 'dialects' of the same language. Translation is more 'difficult': it requires a code book (the genetic code) that converts the nucleic language to that of proteins.

It should be obvious, but note that the promoter is not the same thing as the start codon; nor is the RNA polymerase terminator the stop codon. There are generally untranslated regions (UTRs) at both ends of an RNA transcript, even in bacteria.

Translation requires the cooperation of:

Translation has a low error rate: 10−4 for amino acids, and a speed of about 3 amino acids −1 (eukaryotes) to 20 amino acids s−1 (prokaryotes). These parameters are very similar (if measured per nucleotide) to transcription. Translation occurs on the ribosome.

Ribosomes consist of two subunits: a larger one (the LSU) and a smaller subunit (the SSU). Both subunits contain RNA (the larger contains two or three, the smaller just one) and large numbers of proteins (which are mostly structural). The large subunit contains the active site for peptide bond formation, which is a ribozyme (catalytically active RNA) called peptidyl transferase.

A, P and E sites.

The small subunit initially binds to mRNA like a latch, and also binds tRNAs at the A(minoacyl), P(eptidyl) and E(xit) sites. It controls information flow, whilst the large subunit controls the chemistry. Both help form the groove in which mRNA binds. The E, P and A sites are formed by both ribosomal subunits: the tRNAs interact with mRNA in the SSU P and A sites, whilst the synthesis of protein occurs in the P and A sites of the LSU.

70S ribosome - click for Jmol version
70S ribosome showing A, P and E sites.

Like transcription, translation may be divided into the processes of initiation, elongation and termination. Each step requires the action of several protein factors, RNAs and GTP. It also requires the 'forgotten' part of translation, the aminoacyl tRNA synthetases:

Aminoacyl tRNA synthetases answer the question: "How is tRNA associated with the correct amino acid?" The synthetase selects tRNA by its codon and/or other bases, and attaches the correct amino acid. There are usually just 20 synthetases, many of which must be able to recognise more than one tRNA, so they cannot discriminate purely on the basis of the anticodon.

Amino acid + ATP → Aminoacyl-AMP + PPi.

Aminoacyl-AMP + tRNA → Aminoacyl-tRNA + AMP.

Aminoacyl tRNA synthetase bound to isoleucine tRNA.

Initiation

Initiation in eukaryotes starts with the formation of the eukaryotic 43S pre-initiation complex:

43S pre-initiation complex.

The fourth initiation factor, eIF4A, which is a helicase, unwinds any hairpin loops in the mRNA, exposing the start codon (AUG).

After eIF4A unwinds the mRNA, the 43S pre-initiation complex can bind and find the start codon.

The 43S complex binds to the mRNA and finds the first AUG and eIF2 then hydrolyses GTP, releasing the initiation factors and allowing the LSU to bind and form the eukaryotic 80S initiation complex.

The eIFs are released and the LSU binds to for the 80S initiation complex.

Some mRNAs require additional factors to ensure correct expression at the ribosome: in picornaviruses (such as polio and hepatitis) the VPG protein binds the 5′ end (which is not capped) and directs the ribosome to the correct AUG start codon.

In bacteria, IF1, IF2 and IF3 play similar roles to eIF1, eIF2 and eIF3, and tRNAifmet is always the first tRNA. In both eukaryotes and prokaryotes, the special methionine is usually cleaved off the final polypeptide product. Furthermore, in bacteria, polycistronic mRNAs (which have several start codons) have Shine-Dalgarno sequences c. 5 nt to 5′ of the AUG start codons. This binds the anti-Shine Dalgarno sequence at the end of the 16S SSU rRNA, ensures that the ribosome binds at the correct place(s), not at an out-of-phase codon or an internal methionine.

mRNA: 5′-----AGGAGG-----AUG----3′

rRNA: 3′-…-auUCCUCCacuag---5′

Elongation

The A and P sites are so close on the ribosome that tRNAs must fit contiguous codons.

tRNAs are brought in by EF-Tu (bacteria) or EF-1 (eukaryotes), which hydrolyses GTP in so doing.

EF-Tu brings in charged tRNAs to the A site.

The second elongation factor, EF-G (EF-2), shunts the ribosome along, again using GTP.

EF-G causes conformational changes in the ribosome that shunt it along the mRNA.

This movement brings amino acyl and peptidyl groups together in the active site, catalysing peptide bond formation, and the transfer of the peptidyl group from one tRNA to the next.

Conformational changes in the ribosome cause peptide bond formation.

The de-charged tRNA diffuses out of the ribosome via the E site. Note that the nascent polypeptide chain is always covalently attached to the acceptor stem of the tRNA in the P site.

Decharged tRNA leaves the ribosome.

Peptide bond formation is performed by peptidyl transferase, whose active site contains an adenine ring from the 28S rRNA in large subunit. This performs and acid/base catalysis, just like histidine does in many enzymes. The antibiotic puromycin inhibits ribosomes by mimicking tRNAtyr and preventing the normal transfer of the nascent peptide chain from the tRNA in the P site transfers onto the tRNA in the A site.

Adenine.    Histidine.
Adenine and histidine: note that both have a very similar 5-membered ring (imidazole).

Termination

Stop codons are bound by cytoplasmic release factors, which add water to tRNApeptidyl, cleaving off the polypeptide.

Release factors hydrolyse the peptidyl tRNA to release the polypeptide.

There are two release factors in prokaryotes (RF1 and RF2), but just one in eukaryotes (eRF1), which I think must be the only case where eukaryotes have evolved a simple, elegant system ☺ RFs mimic tRNA in shape, but are made of protein, not RNA.

Prokaryotes also have a special release factor called tmRNA, which provides a template for stalled ribosomes (ones that have stopped translating without meeting a stop codon, maybe because mRNA has been released too early by RNA polymerase). The tmRNA contains a template for an 11 amino acid tag, which marks these abortive proteins for destruction.

Two of the eIF4 proteins (of which there are several besides the IF4A helicase), eIF4E and eIF4G bind the mRNA cap to the PABP on the tail. This means that ribosomes form coiled structures on bound mRNA. Something similar also occurs in prokaryotes. These 'polyribosomes' increase the efficiency of translation, since less mRNA is needed (the ribosomes are only 80 nt apart, so many can simultaneously translate a single mRNA), and also allow efficient re-recruitment of terminated ribosomes back onto the RBS.

Polyribosomes translating a single looped mRNA.

Many antibiotics work on the ribosome or other aspects of transcription or translation:

 

Prokaryote

Eukaryote

mRNA synthesis

Actinomycin-D

Rifampicin

Actinomycin-D

α-Amanitin

Binding tRNA

Tetracycline

 

Initiation complex

Streptomycin

 

Peptidyl transferase

Chloramphenicol

Anisomycin

Translocation

Erythromycin

Cycloheximide

Premature release

Puromycin

Puromycin

This page follows on directly to protein targeting, which discusses where proteins go once they leave the ribosome.

Summary

Test yourself

  1. What processes ensure the accuracy of protein synthesis?
  2. Why does translation require GTP?

Answers

Bibliography

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