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.

A possible mechanism of peptide bond formation on ribosome without mediation of peptidyl transferase.

Ribosome, the ubiquitous organelle, is the site for protein synthesis in all types of cells. The consecutive peptide bonds are formed by the transpeptidation reaction between carboxyl group of peptidyl moiety and the amino group of the aminoacyl moiety. Both the moieties are attached to the appropiate tRNAs positioned on the ribosome at P and A sites, respectively, through codon-anticodon recognition directed by messenger RNA. The reaction seems to proceed by the nucleophillic attack of the amino group of the aminoacyl tRNA at the A site and on the carboxyl of the ester group of the tRNA at P-site of ribosome. The configuration of the carbon atom of the tetrahedral intermediate may be R or S depending on the direction of the nucleophillic attack. After selecting the favorable conformation of this tetrahedral intermediate quantum mechanical calculations have been carried out to determine the energy needed for its formation. A cyclic intermediate where 2'-OH of the ribose sugar of the P-site tRNA is a member of the ring can be formed from the tetrahedral intermediate. This cyclic intermediate produces a free tRNA and a tRNA attached to a planar peptide unit. Analysis of the energetics using semiempirical method for the formation of a cyclic intermediate indicates that the peptide bond formation through the tetrahedral intermediate in S configuration may not need assistance from any outside agent like an enzyme

Sites of ribosomal RNAs involved in the subunit association of tight and loose couple ribosomes.

It was demonstrated previously by kethoxal treatment studies that tight (TC) and loose (LC) couple ribosomes use different sites of the 16 S and 23 S RNAs for subunit association (Burma, D. P., Srivastava, A. K., Srivastava, S., and Dash, D. (1985) J. Biol. Chem. 260, 10517-10525). To localize these sites, a number of oligodeoxynucleotides complementary to the suspected sites of the 16 S and 23 S RNAs were synthesized, and their binding to ribosomes and effects on subunit association were studied. Some of the probes inhibit the association of both TC and LC ribosomes, some inhibit the association of TC but not LC ribosomes, and some inhibit the association of LC but not TC ribosomes. It appears that both TC and LC ribosomes use one common site of association, bases 818-823 of the 16 S and 2308-2313 of the 23 S RNA. The second site, 788-793 of the 16 S RNA and 2753-2758 of the 23 S RNA for TC ribosomes, and 783-791 of the 16 S RNA and 2295-2303 of the 23 S RNA for LC ribosomes, is not shared. This suggests a spatial movement of the ribosomal subunits with respect to one another and lends further support to the model of translocation based on the interconversion of TC and LC ribosomes.

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.

Interconversion of tight and loose couple 50 S ribosomes and translocation in protein synthesis.

On incubation of 50 S ribosomes, isolated from either tight couple (TC) or loose couple (LC) 70 S ribosomes, with elongation factor G (EG-G) and guanosine 5'-triphosphate, a mixture of TC and LC 50 S ribosomes is formed. There is almost complete conversion of LC 50 S ribosomes to TC 50 S ribosomes on treatment with EF-G, GTP, and fusidic acid. Similarly, TC 50 S ribosomes are converted to LC 50 S ribosomes, although partially, by treatment with EF-G and a GTP analogue like guanyl-5'-yl methylenediphosphate (GMP-P(CH2)P) or guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) and including a polymer of 5'-uridylic acid (poly(U] in the incubation mixture. Furthermore, LC 23 S RNA isolated from LC 50 S ribosomes is converted to TC 23 S RNA on heat treatment, but similar treatment does not affect TC 23 S RNA. The interconversion was followed by several physical and biological characteristics of TC and LC 50 S ribosomes, like association capacities with 30 S ribosomes before and after kethoxal treatment, susceptibility to RNase I and polyphenylalanine-synthesizing capacity in association with 30 S ribosomes, as well as thermal denaturation profiles, circular dichroic spectra, and association capacity of isolated 23 S RNAs. These data strongly support the proposition that TC and LC 50 S ribosomes are the products of translocation during protein synthesis. The conformational change of 23 S RNA induced by EF-G and GTP is most probably responsible for the interconversion, and L7/L12 proteins play an important role in the process. A two-site model based on kethoxal data has also been proposed to explain the tightness and looseness of 70 S couples.

Conformational change of 50 S ribosomes on enzymatic binding of phenylalanyl-tRNA.

Tight couple 70 S ribosomes are converted to loose couple ones on enzymatic binding of phenylalanyl-tRNA. Enzymatic binding at 0 degree C as well as nonenzymatic binding does not lead to any change. Further, no change takes place when the P site is occupied by N-acetylphenylalanyl-tRNA. Loose couple 70 S ribosomes are not affected by either enzymatic or nonenzymatic binding of phenylalanyl-tRNA.

Ribosomal activity of the 16 S.23 S RNA complex.

It has been demonstrated in this laboratory that 16 S and 23 S RNAs form a binary complex like 30 S and 50 S ribosomes under certain specific conditions, and 5 S RNA can be incorporated into the complex in stoichiometric amounts in presence of three ribosomal proteins, L5, L18, and L15/25. These studies raised the basic question of whether such complex will have biological activity. Therefore, the following steps in protein synthesis were examined with the complex in place of the ribosomes: (i) poly-U-dependent binding of phenylalanyl tRNA; (ii) EF-G-dependent GTPase activity; (iii) initiation complex formation; (iv) peptidyl transferase activity; and (v) poly-U-dependent polyphenylalanine synthesis. All the steps could be unequivocally demonstrated by the addition of a limited number of proteins although the complex had comparatively much less activity than 70 S ribosomes. It appears that rRNAs are directly involved in various steps of protein synthesis. Furthermore, the 16 S.23 S RNA complex might have acted as a primitive ribosome, as suggested by Crick and Orgel.

Differences in physical and biological properties of 50S ribosomes and 23S RNAs derived from tight and loose couple 70S ribosomes.

Tight couple (TC) 50S ribosomes on treatment with kethoxal lose their capacity to associate with 30S ribosomes whereas loose couple (LC) 50S ribosomes on such treatment fully retain their association capacity. The same is true for 23S RNAs isolated from treated 50S ribosomes or isolated 23S RNAs directly treated with kethoxal, so far as their capacity to associate with 16S RNA is concerned. At certain Mg++ concentrations TC 23S RNA is highly susceptible to the nucleolytic action of single-strand specific enzyme RNase I; LC 23S RNA is quite resistant. The Mg++-dependencies of the two species of 23S RNAs for association with 16S RNA are also quite different. The fluorescence enhancement of ethidium bromide due to binding to TC 23S RNA is slightly less than LC 23S RNA. The hyperchromicity of LC 23S RNA due to thermal denaturation is somewhat more than TC 23S RNA. LC 23S RNA has slightly more elliptic CD spectrum than TC 23S RNA. These results clearly show that 23S RNAs present in TC and LC 50S ribosomes are distinct from each other. It has been recently demonstrated in this laboratory that they can be interconverted by the agents involved in translocation and thus appear to be conformomers.

Association of 16S and 23S ribosomal RNAs to form a bimolecular complex.

Association of the 30S and 50S subunits to generate the 70S ribosomes of Escherichia coli has long been known but the mechanism of this interaction remains obscure. Light-scattering studies indicate that naked 16S and 23S RNAs can also associate under conditions similar to those required for the assembly of ribosomes from the constituent RNAs and proteins. The RNA-RNA association also takes place in the presence of ethanol, which promotes folding of 16S and 23S RNAs into specific compact structures with the morphological features of 30S and 50S ribosomes, respectively. Equimolar amounts of the two RNAs are involved in the association. The formation of a stoichiometric complex was shown by light scattering, sucrose density gradient centrifugation, and composite polyacrylamide/agarose gel electrophoresis. The presence of the two species of RNA in the complex was also shown by gel electrophoresis. The association of naked 16S and 23S RNAs suggests that RNA-RNA interaction may play an important role in the association of 30S and 50S subunits.

Incorporation of 5S RNA into 16S-23S RNA complex.

5S RNA as such is not incorporated into 16S-23S RNA complex formed under reconstitution condition. However, the addition of 50S ribosomal proteins, L5, L18 and L25/L15 results in its incorporation in stoichiometric amount. None of the proteins added individually is capable of incorporating 5S RNA into the complex. Of the different combinations in pairs that are possible out of the four proteins, the pairs L5, L18 and L15, L18 stimulate the incorporation to some extent. Of the four possible triplets, L5, L18, L25 or L5, L15, L18 is the most efficient for maximum incorporation of 5S RNA. The presence of all the four proteins is no more effective than the combinations of the three.

Digoxin toxicity and electrolytes: a correlative study.

Serum levels of sodium, potassium, calcium, magnesium and digoxin were studied in 67 patients on maintenance dose of digoxin, 42 with digitoxicity and 25 without. The mean serum digoxin level of toxic group was significantly higher (p less than 0.001) than non-toxic group. The mean serum potassium was significantly lower in toxic group (p less than 0.05) as compared to the non-toxic group. Of the toxic patients, 23.8% had hypokalemia. Hypokalemia resulted by significantly higher (p less than 0.005) dose of diuretic used in toxic group. The mean serum digoxin level of hypokalemic toxic group was significantly lower as compared to the normokalemic toxic group (p less than 0.001) and it was interesting to note that all hypokalemic toxic patients had their serum digoxin levels below 3 ng/ml (3.84 n mol/ml) and well within therapeutic range. There was a positive correlation between serum digoxin and potassium level amongst toxic patients (p less than 0.001). Thus, in patients on maintenance dose of digoxin therapy, use of large dosage of diuretics may result in hypokalemia, causing digitalis toxicity even at low serum digoxin levels. Serum digoxin level alone may fail as an independent guide in diagnosis of digoxin toxicity in presence of hypokalemia.