• In bacteria protein synthesis begins by the association of one 30S subunit (not the 70S ribosome), one mRNA molecule, a charged tRNAfMet,, three proteins known as initiation factors and guanosine 5’-triphosphate (GTP). These molecules comprise the 30S preinitiation complex. Following formation of the 30S preinitiation complex, a 50S subunit joins to the 30S subunit to form a 70S initiation complex.

    • This joining process requires hydrolysis of the GTP contained in the 30S preinitiation complex. There are two tRNA binding sites which overlap the 30S and 50S subunits. These sites are called as aminoacyl or A site and the peptide or P site; each site consists of a collection of segments of S and L proteins and 23S rRNA. The 50S subunit is positioned in the 70S initiation complex such that the tRNAfMet, which was previously bound to the 30S preinitiation complex, occupies the P site of the 50S subunit. Positioning tRNAfMet, in the P site fixes the position of the anticodon of tRNAfMet, such that it can pair with the initiator codon in the mRNA. Thus, the reading frame is unambiguously defined upon completion of the 70S initiation complex.
    • The A site of the 70S initiation complex is available to any tRNA molecule whose antiocodon can pair with the codon adjacent to the initiation codon. However, entry to the A site by the tRNA requires a helper protein called an elongation factor (EF), specifically EF-Tu. After occupation of the A site a peptide bond between fMet and the adjacent amino acid can be formed.

    • Once it was thought that the blockage of NH2 group of fMet by the formyl group was responsible for peptide bond formation between the COOH group of fMet and the NH2 group of the adjacent amino acid. However, in eukaryotes the starting amino acid is Met and not fMet and protein synthesis proceeds in the correct direction. Presumably, the relative orientation of the two amino acids in the A and P sites determines the linkage that is made.
    • The peptide bond is formed by an enzyme complex called peptidyl transferase. The active site of peptidyl transferase consists of portions of several proteins of the 50S subunit. As the peptide bond is formed, fMet is cleaved from the tRNAfMet, in the P site. After the peptide bond forms, an unchanged tRNA occupies the P site and a dipeptidyl-tRNA is in the A site.
    • At this point three movements, which together comprise the translation step, occur:
    1. The deacylated tRNAfMet, leaves the P site.
    2. The peptidyl-tRNA moves from the A site to the P site and
    3. The mRNA moves a distance of three bases in order to position the next codon at the A site.

    • The translocation step requires the presence of another elongation protein EF-G and hydrolysis of GTP. The movement of the mRNA by three bases is probably dependant on the movement of tRNA from the A site to the P site and in fact, it is likely that mRNA translocation is a consequence of tRNA motion.

    • After translocation has occurred, the A site is again available to accept a charged tRNA molecule having a correct anticodon. If a tRNAMet, molecule, were to enter the A site (because an internal AUG site were present), protein synthesis would stop because a peptide bond cannot form with the blocked NH2 group of fMet. However, in as much as the factor EF-Tu is needed to facilitate tRNA entry into the A site, this misadventure is prevented, since EF-Tu cannot bind to tRNAfMet,.

    • When a chain termination codon is reached, there is no aminoacyl-tRNA that can fill the A site and chain elongation stops. However, the polypeptide chain is still attached to the tRNA occupying P site. Release of the protein is accomplished by release factors (RF), proteins that in part respond to chain termination codons. There are two such release factors in E. coli – RF1, which recognizes the UAA and UAG codons and RF2, which recognizes UAA and UGA. Why the number of release factor is not one (ie. useful to all 3 codons) or three (ie., one for each stop codon) is not known (in eukaryotes, there is only one release factor).

    • Each release factor forms an activated complex with GTP; this complex binds to a termination codon and alters the specificity of peptidyl transferase. In the presence of release factors peptidyl transferase catalyses the reaction of the bound peptidyl moiety with water rather than with the free aminoacyl-tRNA. Thus the polypeptide chain, which has been held in the ribosome solely by the interaction with the tRNA in the P site, is released from the ribosome. The 70S ribosome dissociates into 30S and 50S subunits and the system is ready to start synthesis of a second chain.

Last modified: Thursday, 29 March 2012, 5:18 PM