Peptidyl transferase activity of tRNA: a quantum chemical study.

The mechanism of protein synthesis is still unknown due to inability to detect the so-called enzyme "peptidyl transferase" even after elucidation of high-resolution crystal structure of ribosome. We have recently shown by model building and semi-empirical energy calculation that the tRNA molecule at P-site of ribosome may act as peptidyl transferase (Das et al. (1999) J. Theor. Biol. 200, 193-205). We proposed that the tetrahedral intermediate formed from nucleophylic attack of CO of P-site amino-acylated tRNA by NH2 of A-site amino-acylated tRNA is converted to a six-member ring intermediate by conformational change. This ring intermediate produces a free tRNA and a tRNA covalently linked to a peptide. However, energy of the six-member ring intermediate was calculated to be quite high. We show here that the energy values of all the reactants, intermediates and products are within the expected range when they are calculated using high level ab initio quantum chemical methods.

Mechanism of ribosomal subunit association: oligodeoxynucleotides as probes.

From the kethoxal treatment data [Herr, W.; Chapman, N.M.; Noller, H.F. (1979) J. Mol. Biol. 130, 433-439] some regions of ribosomal RNAs are thought to be responsible for the association of 30S and 50S ribosomes of E. coli to form 70S ribosomes. In order to test this possibility about a dozen oligodeoxynucleotides complementary to the suspected regions of rRNAs were synthesised. Their association with ribosomes and naked rRNAs was tested by the gel filtration technique. In order to check the effects on the ribosomal subunit association or rRNA association either intact 30S and 50S ribosomes or naked 16S and 23S rRNAs were preincubated with the individual oligodeoxynucleotide and its effect was checked by density gradient centrifugation followed by UV absorbance monitoring. Some oligodeoxynucleotides interfered with either subunit association or 16S RNA and 23S RNA association, some with both. These data clearly indicate that RNA-RNA interaction plays the major role in ribosomal subunit association.

Polyclonal antibodies as probes to distinguish between tight and loose couple 50S ribosomes of Escherichia coli.

Antibody has been raised in rabbit against L7/L12 protein of E. coli 50S ribosomes and purified, finally through affinity column. A sensitive assay method using ELISA technique has also been standardised. LC 50S ribosomes react more with the antibody than TC 50S ribosomes. This supports the earlier physical data [Burma D P, Srivastava A K, Srivastava S, Tewari D S, Dash D & Sengupta S K, (1984), Biochem Biophys Res Commun, 124, 970] indicating that L7/L12 stalk region is protruded in medium in LC ribosomes and folded towards the body in TC ribosomes.

Do ribosomal RNAs act merely as scaffold for ribosomal proteins.

Investigations that are being carried out in various laboratories including ours clearly provide the answer which is in the negative. Only the direct evidences obtained in this laboratory will be presented and discussed. It has been unequivocally shown that the interaction between 16S and 23S RNAs plays the primary role in the association of ribosomal subunits. Further, 23S RNA is responsible for the binding of 5S RNA to 16S.23S RNA complex with the help of three ribosomal proteins, L5, L18, L15/L25. The 16S.23S RNA complex is also capable of carrying out the following ribosomal functions, although to small but significant extents, with the help of a very limited number of ribosomal proteins and the factors involved in protein synthesis: (a) poly U-binding, (b) poly U-dependent binding of phenylalanyl tRNA, (c) EF-G-dependent GTPase activity, (d) initiation complex formation, (e) peptidyl transferase activity (puromycin reaction) and (f) polyphenylalanine synthesis. These results clearly indicate the direct involvement of rRNAs in the various steps of protein synthesis. Very recently it has been demonstrated that the conformational change of 23S RNA is responsible for the translocation of peptidyl tRNA from the aminoacyl (A) site to the peptidyl (Ρ) site. A model has been proposed for translocation on the basis of direct experimental evidences. The new concept that ribosomal RNAs are the functional components in ribosomes and proteins act as control switches may eventually turn out to be noncontroversial.

Conformational change of 23S RNA in 50S ribosome is responsible for translocation in protein synthesis.

Since the recognition of the ‘translocation’ phenomenon during protein synthesis several theories have been proposed, without much success, to explain the translocation of peptidyl tRNA from the aminoacyl site to the peptidyl site. The involvement of L7/L12 proteins and therefore the L7/L12 stalk region of 50S ribosomes in the translocation process has been widely accepted. The mobility of the stalk region, as recognised by many workers, must be of physiological significance. It has recently been shown in this laboratory that 50S ribosomes derived from tight and loose couple 70S ribosomes differ markedly in quite a few physical and biological properties and it appears that these differences are due to the different conformations of 23S RNAs. It has also been possible to interconvert tight and loose couple 50S ribosomes with the help of the agents, elongation factor –G, GTP (and its analogues) which are responsible for translocation. Thus loose couple 70S ribosomes so long thought to be inactive ribosomes are actually products of translocation. Further, the conformational change of 23S RNA appears to be responsible for the interconversion of tight and loose couple 50S ribosomes and thus the process of translocation. A model has been proposed for translocation on the basis of the direct experimental evidences obtained in this laboratory.

Presence of precursor ribosome in the ribosomal preparation from chloramphenicol-treated Escherichia coli ΑΒ301/Ι05 (RNase III – ).

On sucrose gradient centrifugation, the ribosomal preparation from chloramphenicoltreated 32P labelled Escherichia coli AB301/105 (RNase III¯ ) showed the presence of a radioactive peak moving slower than the 70S ribosome; this peak disappeared on treatment with RNase III. The presence of precursor 30S RNA was shown in such preparations by affinity chromatography on a lysine-sepharose 4B column as well as polyacrylamide gel electrophoresis. Dialysis against low Mg2± concentration followed by sucrose density gradient electrophoresis. Dialysis against dissociation of 70S ribosome into its subunits, did not lead to the dissociation of the precursor ribosome. However, the dissociation took place upon treatment with RNase III. A tentative model of coupled rRNA transcription and ribosome assembly has been presented.

Immunoprecipitation of 70S, 50S and 30S ribosomes of Escherichia coli.

Antibodies were raised in rabbits against 70S ribosomes, 50S and 30S ribosomal subunits individually. Purified immunoglobulins from the antiserum against each of the above ribosomal entities were tested for their capabilities of precipitating 70S, 50S and 30S ribosomes. The observations revealed the following: (i) The antiserum (IgG) raised against 70S ribosomes precipitates 70S ribosomes completely, while partial precipitation is seen with the subunits, the extent of precipitation being more with the 50S subunits than with 30S subunits; addition of 50S subunits to the 30S subunits facilitates the precipitation of 30S subunits by the antibody against 70S ribosomes. (ii) Antiserum against 50S subunits has the ability to immunoprecipitate both 50S and 70S ribosomes to an equal extent. (iii) Antiserum against 30S subunits also has the property of precipitating both 30S and 70S ribosomes. The differences in the structural organisation of the two subunits may account for the differences in their immunoprecipitability.

Effects of ethidium bromide and berenil on protein synthesis

The effects of ethidium bromide, an intercalating dye and berenil, a nonintercalating dye on the biological activities of Escherichia coli ribosomes have been studied. Ethidium bromide treatment drastically reduced both enzymatic and nonenzymatic initiation complex formation, enzymatic as well as nonenzymatic binding of phenylalanyl tRNA, peptidyl transferase, GTPase as well as the overall protein synthesising activity as measured by the poly U-dependent polymerization of phenylalanine. On berenil treatment, however, only enzymatic formation of the initiation complex is marginally reduced. Other reactions are not markedly affected except the enzymatic phenylalanyl tRNA binding which is slightly decreased only at high Mg2+ concentration; the treated ribosome has lowered polymerizing activity at sub-optimal Mg2+ concentration (10 mM). Although it has already been shown in this laboratory that treatment with either dye leads to the unfolding of the structure of the ribosome, the present studies indicate that berenil treatment does not alter the structure of the ribosome drastically in contrast to ethidium bromide treatment.

Alteration of the structure of the Escherichia coli ribosomes on treatment with Fab fragment of immunoglobulin raised against the ribosomes.

Rabbits were immunised against Escherichia coli ribosomes and the partially purified immunoglobulin G fraction had maximum ability to precipitate the ribosomes as well as the extracted ribosomal proteins. By digestion of immunoglobulin G with papain, monovalent Fab fragments were produced. The 70 S ribosome and its subunits (50 S and 30 S) were separately treated with Fab and then tested in the kinetic assay of degradation of ribosomes by ribonuclease I at various Mg2+ concentrations. Treated ribosomes and their subunits were degraded at faster rates than the nontreated ones; the rates in both the control and the treated cases were dependent on the concentration of Mg2+. These results indicate the unfolding of the structure of the ribosome on treatment with antibody fragments, which may be due to the weakening of the interaction between rRNAs and ribosomal proteins.