Initiation of translation in prokaryotes involves the assembly of the components of the translation system which are: the two ribosomal subunits, the mRNA to be translated, the first aminoacyl tRNA (the tRNA charged with the first amino acid), GTP (as a source of energy), and initiation factors which help the assembly of the initiation complex. Prokaryotic initiation results in the association of the small and large ribosomal subunits and binding of first aminoacyl tRNA (fmet-tRNA) through anticodon-codon base pairing with the initiation codon of mRNA.
The ribosome consists of three sites: the A site, the P site, and the E site. The A site is the point of entry for the aminoacyl tRNA (except for the first aminoacyl tRNA, fmet-tRNA, which enters at the P site). The P site is where the peptidyl tRNA is formed in the ribosome. And the E site which is the exit site of the now uncharged tRNA after it gives its amino acid to the growing peptide chain.
Initiation of translation begins with the 50s and 30s ribosomal subunits dissociated. IF1 (initiation factor 1) blocks the A site to insure that the fMet-tRNA can bind only to the P site and that no other aminoacyl-tRNA can bind in the A site during initiation, while IF3 blocks the E site and prevents the two subunits from associating. IF-2 is a small GTPase which binds fmet-tRNA and helps its binding with the small ribosomal subunit. The 16s rRNA of the small 30S ribosomal subunit recognizes the ribosomal binding site on mRNA (the Shine-Dalgarno sequence, 5-10 base pairs upstream of the start codon(AUG)) The Shine-Dalgarno sequence is found only in prokaryotes. This helps to correctly position the ribosome onto the mRNA so that the P site is directly on the AUG initiation codon. IF-3 helps to position fmet-tRNA into the P site, such that fmet-tRNA interacts via base pairing with the mRNA initiation codon (AUG). Initiation ends as the large ribosomal subunit joins the complex causing the dissociation of initiation factors. Note that prokaryotes can differentiate between a normal AUG (coding for methionine) and an AUG initiation codon (coding for formylmethionine and indicating the start of a new translation process).
Elongation of the polypeptide chain involves addition of amino acids to the carboxyl end of the growing chain. The growing protein exits the ribosome through the polypeptide exit tunnel in the large subunit.
Elongation starts when the fmet-tRNA enters the P site, causing a conformational change which opens the A site for the new aminoacyl-tRNA to bind. This binding is facilitated by elongation factor-Tu (EF-Tu), a small GTPase. Now the P site contains the beginning of the peptide chain of the protein to be encoded and the A site has the next aminoacid to be added to the peptide chain. The growing polypeptide connected to the tRNA in the P site is detached from the tRNA in the P site and a peptide bond is formed between the last amino acids of the polypeptide and the amino acid still attached to the tRNA in the A site. This process, known as peptide bond formation, is catalyzed by a ribozyme, peptidyltransferase, an activity intrinsic to the 23S rRNA in the 50s ribosomal subunit. Now, the A site has newly formed peptide, while the P site has an unloaded tRNA (tRNA with no amino acids). In the final stage of elongation, translocation, the ribosome moves 3 nucleotides towards the 3'end of mRNA. Since tRNAs are linked to mRNA by codon-anticodon base-pairing, tRNAs move relative to the ribosome taking the nascent polypeptide from the A site to the P site and moving the uncharged tRNA to the E exit site. This process is catalyzed by elongation factor G (EF-G).
The ribosome continues to translate the remaining codons on the mRNA as more aminoacyl-tRNA bind to the A site, until the ribosome reaches a stop codon on mRNA(UAA, UGA, or UAG).
Termination occurs when one of the three termination codons moves into the A site. These codons are not recognized by any tRNAs. Instead, they are recognized by proteins called release factors, namely RF1 (recognizing the UAA and UAG stop codons) or RF2 (recognizing the UAA and UGA stop codons). A third release factor RF-3 catalyzes the release of RF-1 and RF-2 at the end of the termination process. These factors trigger the hydrolysis of the ester bond in peptidyl-tRNA and the release of the newly synthesized protein from the ribosome.
The post-termination complex formed by the end of the termination step consists of mRNA with the termination codon at the A-site, tRNAs and the ribosome. Ribosome recycling step is responsible for the disassembly of the post-termination ribosomal complex. Once the nascent protein is released in termination, Ribosome Recycling Factor and Elongation Factor G (EF-G) function to release mRNA and tRNAs from ribosomes and dissociate the 70s ribosomes into the 30s and 50s subunits. IF-3 also helps the ribosome-recycling process by converting transiently dissociated subunits into stable subunits by binding to the 30S subunits. This "recycles" the ribosomes for additional rounds of translation.
Translation is carried out by more than one ribosome simultaneously. Because of the relatively large size of ribosomes, they can only attach to sites on mRNA 35 nucleotides apart. The complex of one mRNA and a number of ribosomes is called a polysome or polyribosome.
Effect of antibiotics
Several antibiotics exert their action by targeting the translation process in bacteria. They exploit the differences between prokaryotic and eukaryotic translation mechanisms to selectively inhibit protein synthesis in bacteria without affecting the host. Examples include:
- Puromycin has a structure similar to the tyrosinyl aminoacyl-tRNA. Thus, it binds to the ribosomal A site and participates in peptide bond formation, producing peptidyl-puromycin. However, it does not engage in translocation and quickly dissociates from the ribosome causing a premature termination of polypeptide synthesis.
- Streptomycin causes misreading of the genetic code in bacteria at relatively low concentrations and inhibits initiation at higher concentrations, by binding to the 30s ribosomal subunit.
- Other aminoglycosides as Tobramycin and Kanamycin prevent ribosomal association at the end of initiation step and cause misreading of the genetic code.
- Tetracyclines block the A site on the ribosome, preventing the binding of aminoacyl tRNAs.
- Chloramphenicol blocks the peptidyl transfer step of elongation on the 50s ribosomal subunit in both bacteria and mitochondria.
- Macrolides and Lincosamides bind to the 50s ribosomal subunits inhibiting the peptidyltransferase reaction or translocation or both.
- Structure fo the E. coli protein-coducting channel bound to at translating ribosome, K. Mitra, et al. Nature (2005), vol 438, p 318
- Hirokawa et al. (2006) "The Ribosome Recycling Step: Consensus or Controversy?". Trends in Biochemical Sciences Vol. 31(3), 143-149.