As with any of the polymerization reactions, protein synthesis can be divided into three phases:
where a functionally competent ribosome is assembled in the correct place on an mRNA ready to commence protein synthesis.
whereby the correct amino acid is brought to the ribosome, is joined to the nascent polypeptide chain, and the entire assembly moves one position along the mRNA.
which happens when a stop codon is reached, there is no amino acid to be incorporated and the entire assembly dissociates to release the newly-synthesized polypeptide.
There are two rules about protein synthesis to keep in mind:
- mRNA is translated 5' -> 3'
- Proteins are synthesized from the N-terminus to the C-terminus
This account describes the steps of protein synthesis in bacteria; we will mention eukaryotic protein synthesis briefly at the end.
This phase of protein synthesis results in the assembly of a functionally competent ribosome in which an mRNA has been positioned correctly so that its start codon is positioned in the P (peptidyl) site and is paired with the initiator tRNA.
The following ingredients are needed for this phase of protein synthesis:
- Two ribosome subunits - 30S and 50S
- The mRNA
- Three Initiation Factors - IF1, IF2 (GTP) and IF3
- The initiator fMet-tRNAfMet.
The following steps take place:
Binding of the ribosome 30S subunit with Initiation Factors
IF3 promotes the dissociation of the ribosome into its two component subunits. The presence of IF3 permits the assembly of the initiation complex and prevents binding opf the 50S subunit prematurely.
IF1 assists IF3 in some way, perhaps by increasing the dissociation rate of the 30S and 50S subunits of the ribosome.
Binding of tthe mRNA and the fMet-tRNAfMet
IF3 assists the mRNA to bind with the 30S subunit of the ribosome so that the start codon is correctly positioned at the peptidyl site of the ribosome. The mRNA is positioned by means of base-pairing between the 3' end of the 16S rRNA with the Shine-Dalgarno sequence immediately upstream of the start codon.
IF2(GTP) assists the fMet-tRNAfMet to bind to the 30S subunit in the correct site - the P site.
It is not clear whether the mRNA or fMet-tRNAfMet binds first. It may be that either can bind first.
At this stage of assembly, the 30S initiation complex is complete and IF3 can dissociate.
Binding of the ribosome 50S subunit and release of Initiation Factors
As IF3 is released, the 50S subunit of the ribosome binds to complete the initiation complex. Simultaneously, GTP hydrolysis occurs on IF2. This hydrolysis may be helped by the L7/L12 ribosomal proteins rather than by IF2 itself. Hydrolysis is required for dissociation of IF2. GTP hydrolysis probably serves as a timing mechanism to ensure that the tRNA is correctly positioned before IF3 dissociates.
Once IF2 and IF1 are both released, translation can proceed.
Three special Elongation Factors are required for this phase of protein synthesis: EF-Tu (GTP), EF-Ts and EF-G (GTP).
The Elongation phase of protein synthesis consists of a cyclic process whereby a new aminoacyl-tRNA is positioned in the ribosome, the amino acid is transferred to the C-terminus of the growing polypeptide chain, and the the whole assembly moves one position along the ribosome:
Binding of a new aminoacyl-tRNA at the A site
At the start of each cycle, the A (aminoacyl) site on the ribosome is empty, the P (peptidyl) site contains a peptidyl-tRNA, and the E (exit) site contains an uncharged tRNA.
The elongation factor, EF-Tu (GTP) binds with an aminoacyl-tRNA and brings it to the ribosome. Once the correct aminoacyl-tRNA is positioned in the ribosome, GTP is hydrolyzed and EF-Tu (GDP) dissociates away from the ribosome.
There are two ways that EF-Tu functions to ensure that the correct aminoacyl-tRNA is in place:
- EF-Tu prevents the aminoacyl end of the charged tRNA from entering the A site on the ribosome. This ensures that codon-anticodon pairing is checked first before the charged tRNA is irreversibly bound in the A site and a new, potentially incorrect, peptide bond is made.
- GTP hydrolysis is SLOW and EF-Tu cannot dissociate from the ribosome until it occurs. The amount of time prior to GTP hydrolysis allows the final fidelity check to take place.
If the anticodon-codon interaction is incorrect, the aminoacyl-tRNA simply dissociates and a new one is brought in. This check, however, can verify nothing about the aminoacid -- it simply verifies that the correct pairing takes place.
Experiments using GTP analogues have been used to establish these results:
- If a GTP analogue such as GTP-g-S, which is hydrolyzed very slowly, is used then protein synthesis slows down because of the slow rate of hydrolysis but it also becomes more accurate because there is more time to check that the correct aminoacyl-tRNA is in place.
- If a GTP analogue such as GMP-PCP, which contains a non-hydrolyzable methylene bridge between the b and g phosphates, is used then protein synthesis stops because EF-Tu cannot dissociate from the ribosome.
EF-Tu is the most abundant protein in the E. coli cell. There are approximately 70-100,000 molecules/cell which is 5% of the total cell protein. There are also approximately 70-100,000 tRNA molecules/cell. Nearly all of the aminoacyl-tRNA in the cell is bound by EF-Tu.
EF-Tu cannot bind with tRNAfMet. This tRNA has a slight difference in its structure compared with that of tRNAMet which means that it is not bound by EF-Tu.
EF-Tu (GDP) is inactive and cannot function to bind aminoacylated tRNAs. However, EF-Tu has a higher affinity for GDP (Ka = 10-8M) than for GTP (Ka = 10-6M).
In order to recycle EF-Tu, the elongation factor EF-Ts binds to the EF-Tu (GDP) complex to displace the GDP. GTP then, in turn, displaces EF-Ts. Many other G-proteins require a guanine nucleotide release protein (GNRP) to release GDP; EF-Ts is the GNRP for EF-Tu.
Formation of the new peptide bond (Transpeptidation)
Peptide bond formation occurs as a result of nucleophilic attack by the lone pair of electrons on the amino nitrogen of the aminoacyl-tRNA on the carbonyl carbon that attaches the growing polypeptide chain to a tRNA molecule in the P site of the ribosome. As a result, the peptide chain is attached to the tRNA which is paired with the codon in the A site. The new amino acid is, therefore, added to the C-terminal end of the polypeptide chain.
Older illustrations show this reaction as a transfer of the entire polypeptide chain from the tRNA in the P site to the tRNA in the A site. This is not an accurate representation. It is more likely that the aminoacyl arm of the tRNA in the A site extends to join with the polypeptide chain in the P site.
The peptidyltransferase activity of the ribosome which catalyzes this reaction is located on the 23S rRNA though it will be assisted by some of the ribosomal protein subunits. In other words, peptidyl transferase is a ribozyme - another example of a catalytic RNA.
Translocation of the Ribosome
Finally, the ribosome translocates along the mRNA thereby moving the new peptidyl-tRNA to the P site and the old (now uncharged) tRNA, which has just lost its peptidyl chain, to the E site. This step requires the elongation factor, EF-G(GTP). There are 20,000 molecules/cell of EF-G which is the same as the number of ribosomes.
GTP is hydrolyzed during translocation and, once again, GTP hydrolysis is required for dissociation of EF-G not for binding.
EF-G blocks the binding of aminoacyl tRNAs to the A site as well as blocking the binding of Release Factors. It effectively makes sure that translocation must take place before the cycle continues.
EF-G and the tRNA-EF-Tu complex are mutually exclusive. The structures of these two are remarkably similar and demonstrate very nicely why these two cannot bind to the ribosome simultaneously:
The following diagram summarizes the movement of tRNA through the ribosome during the elongation phase of protein synthesis:
A new codon is now positioned at the A site and awaits a new aminoacyl-tRNA.
The final phase of protein synthesis requires that the finished polypeptide chain be detached from a tRNA. This can only happen in response to the signal that a stop codon has been reached. After hydrolysis, the ribosome subunits dissociate.
Binding of Release factors
There are no tRNAs that recognize the stop codons. Rather they are recognized by release factor RF1 (which recognizes the UAA and UAG stop codons) or RF2 (which recognizes the UAA and UGA stop codons). These release factors act at the A site of the ribosome. A third release factor, RF3 (GTP), stimulates the binding of RF1 and RF2.
Hydrolysis of the peptidyl-tRNA
Binding of the release factors alters the peptidyltransferase activity so that water is now the nucleophilic attack agent. The result is hydrolysis of the peptidyl-tRNA and release of the completed polypeptide chain. The uncharged tRNA then dissociates as do the release factors. GTP is hydrolyzed.
Finally, the ribosome dissociates into its 30S and 50S subunits and the mRNA is released. IF3 may help this process.
Antibiotics and Protein Synthesis
Many antibiotics and toxins funtion by blocking certain steps during protein synthesis. As well as their utility in treating infections, antibiotics have been useful in dissecting many of the molecular details of the steps and reactions of protein synthesis. The following will give you a feel for this important topic.
Chloramphenicol Inhibits peptidyl transferase in prokaryotes. It binds near the L16 protein and seems to prevent the aminoacylated end of charged tRNAs from binding correctly to the A site on the ribosome. Cycloheximide Inhibits peptidyl transferase in eukaryotes. Diphtheria Toxin Inhibits the activity of EF-G byADP-ribiosylation. Erythromycin Blocks the translocation step of protein synthesis. Fusidic Acid Blocks the dissociation of eEF-2 during protein synthesis in eukaryotes. Kanamycin Causes misreading of the code by interfering with the wobble base pairing. Kirromycin Blocks dissociation of GDP from EF-Tu after hydrolysis. This prevents dissociation of EF-Tu from the ribosome and effectively stalls protein synthesis. Puromycin
Causes premature chain termination. Its structure resembles that of the 3' end of a tyrosyl-tRNA and it participates as a substrate in a peptidyl transferase reaction.
However, once it is added to the 3' end of a nascent protein, it does not provide a suitable centre for any further nucleophilic reactions, and protein synthesis is aborted.
Streptomycin This antibiotic was the first aminoglycoside characterized. It inhibits prokaryotic ribosomes in a couple of ways. It causes misreading by interfering with the normal pairing between codon and anticodon. It can also prevent initiation. Streptomycin resistant bacteria carry an altered S12 subunit. Tetracycline Inhibits aminoacyl-tRNA binding to the A site on the ribosome.
Protein Synthesis in Eukaryotes
A major difference between eukaryotes and prokaryotes is that, in a typical eukaryotic cell, protein synthesis takes place in the cytoplasm while transcription and RNA processing take place in the nucleus. In bacteria, these two processes can be coupled so that protein synthesis can start even before transcription has finished.
The steps of protein synthesis are basically the same in eukaryotic cells as in prokaryotes. The ingredients, however, can be different -- we have already described some of them.
- Ribosomes are larger. 60S and 40S subunits combine to give 80S ribosomes. They contain 4 rRNAs: 28S, 5.8S and 5S in the 60S subunit; 18S in the 40S subunit.
- While the initiating amino acid in eukaryotic protein synthesis is still methionine, it is not formylated.
- Eukaryotic mRNA is capped. This is used as the recognition feature for ribosome binding -- not the 18S rRNA.
- The initiation phase of protein synthesis requires over 10 eukaryotic Initiation Factors (eIFs) one of which is the cap binding protein.
- The eukaryotic elongation phase closely resembles that in prokaryotes. The corresponding elongation factors are eEF-1a (EF-Tu), eEF-1bg (EF-Ts) and eEF-2 (EF-G).
- Eukaryotes require just a single release factor, eRF.
Coordinating Protein Synthesis with mRNA Synthesis
It has recently been found that the eukaryotic initiation factor eEF-4G binds not only with other factors in the initiation complex but also with PABP (polyA binding protein) which binds to the polyA tail of mRNA.
It is though that the binding of eEF-4G to PABP serves as a crticial recruitment step for driving downstream translation.
In another sense, however, the binding of eEF-4G to PABP represents a mechanism to ensure that only mature intact mRNAs are translated.
A similar problem arises in prokaryotes. Bacterial mRNA turns over much faster than eukaryotic mRNA and there is a much higher probability that the 3'-end of an mRNA will be degraded. If this happens, the consequences could be severe. If an mRNA has lost its stop codons, there will be no signals to promote dissociation of the ribosomes. Any ribosomes that have bound to a defective mRNA will therefore stall when they reach the broken end unable to continue and unable to dissociate efficiently.
E. coli (and other bacteria) has a mechanism to deal with this situation.
E. coli contains a small RNA, encoded by the ssrA gene, is synthesized as a 457 nt precursor RNA that is processed by RNaseE to a mature 363 nt RNA. This RNA is also known as tmRNA or 10Sa RNA.
The ssrA RNA has a number of important properties:
- Its secondary and tertiary structure partially resembles that of tRNA
- It can be charged with alanine
- It can be used as an mRNA which codes for a 10 amino acid long oligopeptide: ANDENYALAA.
The mechanism of action of the ssrA RNA is shown in the following figure:
When a ribosome stalls, the ssrA RNA charged with alanine is brought to the A-site of the ribosome by the SsrB protein. Peptidyl transferase activity transfers the nascent polypeptide to the alanine attached to ssrA.
The mRNA template is also displaced by the ssrA RNA. Further protein synthesis now uses ssrA as a template and ten further amino acids (ANDENYALAA) are added to the C-terminal end of the polypeptide.
However, the final two amino acids that are added (AA) mark the new protein for proteolysis by the two proteases ClpAP and ClpXP.
Thus any proteins that are only partially synthesized by stalled ribosomes can be rapidly destroyed and turned over.