Binding of AA-tRNA at site 'A' of ribosome

Each ribosome has two cavities, in which tRNA can be inserted. These are 'P' site (peptidyl site) and 'A' site (amino acyl site). It is not known whether f-met-tRNA_f^met comes on P site or on A site, but ultimately it has to be present on P site, to make 'A' site available for the next amino acyl tRNA (AA-tRNA).

Another site called 'R' site located on smaller subunit of ribosome, has also been proposed. The presence of 'R' site has been confirmed and its role has been discussed. It is proposed that 'R' site plays a role in the improvement of accuracy of translation. It is postulated that aminoacyl tRNA first binds to R site involving codon-anticodon pairing (Fig. 34.7). Later aminoacyl tRNA is flipped to A site using energy from GTP molecule (Fig. 34.8). During this flipping, tRNA is held only by codon-anticodon pairing. If this pairing is weak due to mismatching of wrong tRNA, aminoacyl tRNA will fly off the ribosome. R site thus provides a correcting measure.

After formation of 70S initiation complex, the first amino acyl tRNA (AA-tRNA) enters 'A' site. Factors responsible for this entry include the elongation factors EF-Tu (u means unstable when heated) and EF-Ts (s means stable when heated). Although AA-tRNA can bind to ribosome even without the help of elongation factors (EF-Tu, EF-Ts), proper binding occurs only in the presence of EF-Tu, which is responsible for placing AA-tRNA on the A site.

The elongation factor EF-Tu first combines with GTP and changes to an active binary complex (EF-Tu-GTP) which binds with AA-tRNA (but it can not bind to f-met-tRNA_f^met) to form a ternary complex (Fig. 34.9).



The ternary complex, AA-tRNA-EF-Tu-GTP binds only to the A site, the P site being already occupied by f-met-tRNA_f^met at the initial stage or by peptidyl tRNA at later stages of elongation. After binding to ribosome, GTP is cleaved to release EF-Tu-GDP+P. EF-Ts displaces GDP and associates with Tu and then GTP can again associate with EF-Tu to start another cycle for the binding of AA-tRNA. EF-Ts is released at this stage to be used again with EF-Tu-GDP complex (Fig. 34.9).



In eukaryotes as typified by red blood cells, there are two elongation factors eEFl and eEF2, the former being considered analogous to EF-T (both EF-Tu and EF-Ts) of E. coli and the latter analogous to EF-G (see later in the section). However, eEFl may have several polypeptides in addition to the two which may be analogous to EF-Tu and EF-Ts. The elongation factors in chloroplasts and mitochondria may be completely independent and have not been characterized so far.

Formation of peptide bond
This is a catalytic reaction during which a peptide bond is formed between the free carboxyl group (-COOH) of the peptidyl tRNA at the 'P' site and the free amino (-NH₂) group present with amino acyl tRNA at the 'A' site. The enzyme involved in this reaction is peptidyl transferase and is an integral part of 50S ribosomal subunit. After the formation of peptide bond, the tRNA at 'P' site is deacylated and the tRNA at 'A' site now carries the polypeptide.

Translocation of peptidyl tRNA from 'A' to 'P' site
The peptidyl tRNA present at 'A' site is now translocated to 'P' site. For translocation of peptidyl tRNA from 'A' site to P site, there are two models available : (i) According to two sites (A, P) model, deacylated tRNA is liberated from 'P' site, and with the help of one GTP molecule and an elongation factor EF-G (called EF-G, since it was isolated as GTP requiring factor; also earlier called transfer factor, TFII or translocase), the peptidyl tRNA is translocated from 'A' to 'P' site (Fig. 34.10). Thus according to this model, tRNA is either entirely in the A site or entirely in the P site, (ii) According to newer three sites (A, P, E) model, initially the aminoacyl end of the t-RNA bound to A-site moves to the P site on 50S subunit at the time of peptide transfer (before translocation), but only later during translocation, the anticodon end of this tRNA moves from A to P site on 30S subunit. Only this later step requires action of elongation factor EF-G. In this model thus, there is an intermediate state, when anticodon end of tRNA is still on A site (on 30S subunit), while the aminoacyl end occupies P site (on 50S subunit see Fig. 34.11). A third tRNA binding site, 'E' (Exit) was also recognized, through which tRNA leaves the ribosome. E site is found mainly on 50S subunit, and deacylated tRNA interacts with it through its CCA sequence at its 3' end. The movement of deacylated tRNA from P to E site also occurs in two steps : the aminoacyl end moves to E site during peptide bond formation, and the anticodon end leaves the P site (on 30S) only during translocation. This three site model suggests that tRNAs interact mainly at their ends. It also assumes that a tRNA remains bound to ribosome at one or the other of its two ends (aminoacyl end, anticodon end), and does not leave ribosome at both its ends simultaneously (either on A or P site). The two step transfer of tRNAs "from A to P site and from P to E site could result from reciprocating motions of the two subunits of a ribosome. It means that SOS and 30S subunits move alternately and not simultaneously.

The requirement of EF-G and GTP for translocation was revealed by the use of antibiotic puromycin, which closely resembles AA-tRNA in structure (Fig. 34.12) and can, therefore, occupy 'A' site and form peptidyl puromycin. The later can not form further peptide bond and is released prematurely causing lethality. This puromycin reaction is dependent on the presence of EF-G and GTP, suggesting their significance in translocation process.

The elongation factor EF-G binds to ribosome and is released on hydrolysis of GTP, which is a ribosomal function. EF-G and EF-Tu can not bind to ribosome simultaneously, so that the entry of a fresh AA-tRNA on 'A' site and the translocation of peptidyl tRNA from 'A' to 'P' site have to follow each other and can not occur simultaneously. For instance, EF-Tu-GDP must be released before EF-G can bind and similarly EF-G should be released before AA-tRNA-EF-Tu-GTP can bind to 'A' site. This was confirmed by the observation that if ribosome-EF-G-GTP complex is stabilized by a steroid antibiotic fusidic acid, so that EF-G is not released after translocation, further process of translation stops, due to inability of AA-tRNA-EF-Tu-GTP complex to enter the 'A' site. This exclusion of one elongation factor by the other allows orderly translation in protein synthesis. The details about EF-Tu, EF-Ts and EF-G are given in Table 34.5.


In eukaryotes, the elongation factor needed for translocation is called eEF-2. Unlike prokaryotes, a stable complex of eEF-2 with GTP can be isolated and this complex can bind to ribosome.

The above account of elongation of polypeptide suggests that for the formation of one peptide bond, one ATP molecule (for activation of amino acid) and two GTP molecules (one for transfer of AA-tRNA to 'A' site and the other for translocation of peptidyl tRNA from 'A' to 'P' site) are required. However, in 1993 evidence has been made available, which suggests that the number of GTP molecules for the formation of one peptide bond can be three on even more. For instance, it is suggested that for the transfer of one AA-tRNA, two molecules of che binary complex, EF-Tu-GTP, may be required, so that the total GTP molecules for one peptide bond will be three (two GTP for transfer of AA-tRNA and one GTP for translocation of peptidyl tRNA).