The elongation factor Tu.GTPase reaction: effect of 2'(3')-O-aminoacyl oligoribonucleotides.

The activity of synthetic (2'(3')-O-aminoacyl trinucleotides, C-C-A-Phe, C-C-U-Phe, C-U-A-Phe, U-C-A-Phe and C-A-A-Phe, in promoting the EF-Tu.70 S ribosome-catalyzed GTP hydrolysis was investigated. It was found that the activity decreases in the order C-C-A-Phe greater than C-U-A-Phe greater than U-C-A-Phe greater than C-A-A-Phe much greater than C-C-U-Phe. Thus, the substitution in 'natural' C-C-A sequence with other nucleobases weakens binding of 2'(3')-O-aminoacyl trinucleotides to EF-Tu, with the substitution at the 3'-position having the most profound effect. Since the 2'(3')-O-aminoacyl oligonucleotides mimic the effect of the aa-tRNA 3'-terminus on EF-Tu.GTPase, it follows that EF-Tu probably directly recognizes structure of nucleobases in the aa-tRNA 3'-terminus, with the 3'-terminal adenine playing the most important role.

Inhibition of the peptidyltransferase acceptor site by 2'(3')-O-cycloleucyl- and alpha-aminoisobutyryl derivatives of cytidylyl-(3'-5')adenosine.

2'(3')-O-(N-Benzyloxycarbonylcycloleucyl)adenosine (1a) was prepared by esterification of 5'-O-(4-methoxytrityl)adenosine with N-benzyloxycarbonylcycloleucine in the presence of dicyclohexylcarbodiimide and subsequent deprotection in acidic medium. The compound 1a was separated into pure 2'- and 3'-isomers using HPLC; these isomers were found to undergo an easy interconversion. Compound 1a was coupled with N-dimethylaminomethylene-2',5'-di-O-tetrahydropyranylcytidine 3'-phosphate in the presence of dicyclohexylcarbodiimide to give, after subsequent deblocking, cytidylyl(3' leads to 5')2'(3')-O-cycloleucyladenosine (1c). Compound 1c, as well as the related cytidylyl(3' leads to 5')2'(3')-O-(alpha-aminoisobutyryl)adenosine (1d), inhibited the peptidyltransferase catalyzed transfer of an AcPhe residue to puromycin in the Ac[14C]Phe-tRNA . poly(U) . 70 S E. coli ribosome system. A half of the maximum inhibition of AcPhe-puromycin formation (at 10(-5) M puromycin) was achieved at 9.5 . 10(-6) M of compound 1c and 9 . 10(-5) M of compound 1d, respectively. The inhibition of the puromycin reaction by compound 1d shows a mixed-type of inhibition kinetics. Further, none of the compounds 1c and 1d was an acceptor in the peptidyltransferase reaction. Both compounds 1c and 1d inhibited the binding of C-A-C-C-A[14C]Phe to the A site of peptidyltransferase in a system containing tRNAPhe . poly(U) . 70 S E. coli ribosomes, in which compound 1d was a much stronger inhibitor than 1c. These results indicate that the derivatives such as compounds 1c and 1d which contain an anomalous amino acid with a substituent in lieu of alpha-hydrogen can interfere with the peptidyltransferase A site; however, they are not acceptors in the peptidyltransferase reaction probably due to a misfit of the alpha-substituent.

Effect of thiostrepton and 3'-terminal fragments of aminoacyl-tRNA on EF-Tu and ribosome-dependent GTP hydrolysis.

The effect of the antibiotics thiostrepton and micrococcin on EF-Tu-catalyzed (ribosome-dependent) GTP hydrolysis in the presence of A-Phe, C-A-Phe, or C-C-A-Phe (related to the sequence of the 3'-terminus of aminoacyl-tRNA)(System I) or by methanol ('uncoupled GTPase', System II) was investigated. In System I, thiostrepton increases the binding affinities of the effectors to the EF-Tu.GTP.70 S ribosome complex, as well as the extent of the GTP hydrolysis, while the KmGTP is virtually unchanged. Similarly, in the uncoupled system (System II) and in the absence of effectors, thiostrepton significantly increases VmaxGTP, whereas KmGTP remains unaffected. Micrococcin is without any effect in both systems. The 'uncoupled GTPase' (in System II) is also strongly inhibited by C-A-Phe. The results indicate the crucial role of the EF-Tu site which binds the aminoacylated C-C-A terminus of aminoacyl-tRNA in promoting GTP hydrolysis. It follows that the binding of the model effectors (such as C-C-A-Phe) to that site is favorably influenced by the interaction of thiostrepton with the 50 S ribosomal subunit, whereas thiostrepton, per se, does not influence the affinity of EF-Tu for GTP.

The peptidyltransferase center of Escherichia coli ribosomes: binding sites for the cytidine 3'-phosphate residues of the aminoacyl-tRNA 3'-terminus and the interrelationships between the acceptor and donor sites.

The substrate specificity of the acceptor site of peptidyltransferase of Escherichia coli 70 S ribosomes was investigated in Ac-Phe-tRNA . poly(U) . 70 S ribosome (system A) and tRNC-A-Phe . poly(U) . C-A-C-C-A-Phe . 70 S ribosome (system B) systems by using C-C-A-Gly, C-C-A-Phe, C-A-Gly and C-A-Phe as analogs of the 3'-terminus of aminoacyl-tRNA. It was found that an addition of CP residue to C-A-Gly and C-APhe resulted in an increase of the acceptor activity in system A; the increase is more remarkable for C-A-Gly than for C-A-Phe, while the acceptor activities of C-C-A-Gly and C-C-A-Phe are roughly similar. On the other hand, dramatically increased binding affinities of C-C-A-Phe and C-C-A-Gly relative to C-A-Phe and C-A-Gly for the A site of peptidyltransferase were observed in system B using an inhibition assay; C-C-A-Phe binds much more strongly than C-C-A-Gly. The results indicate the important role of the third CP residue and the aminoacyl moiety of the 3'-terminus of aminoacyl-tRNA in the interaction with the acceptor site of peptidyltransferase, as well as the existence of cooperative effects between A and P sites of peptidyltransferase. These effects, depending on an occupancy of P site, may significant the specificity of the peptidyltransferase A site.

Interaction of elongation factor Tu with 2'(3')-O-aminoacyloligonucleotides derived from the 3' terminus of aminoacyl-tRNA.

The interaction between Escherichia coli elongation factor-Tu-GTP complex and chemically synthesized 2'(3')-O-aminoacyldinucleoside phosphates with the nucleotide sequence of the 3' terminus of aminoacyl-tRNA (AA-tRNA) has been studied. It was found that C-A-Phe, C-A-Pro, and C-A-Asp interact with EF-Tu-GTP, causing the release of GTP bound to the enzyme. The specificity of this interaction closely resembles that of AA-tRNA since C-A and C-A(Ac-Phe) as well as the corresponding tRNAs are inactive. The 3'-O-aminoacyl derivative C-2'-dA-Phe does not interact with EF-Tu-GTP, whereas the 2'-O-aminoacyl derivative C-3'-dA-Phe is almost as active as the 2'(3')-O-aminoacyl derivative, C-A-Phe. C-A-Phe also interacts with the EF-Tu-GDP complex in a manner similar to its interaction with EF-Tu-GTP. It is concluded that interaction of 2'(3')-O-aminoacyloligonucleotides possessing the sequence of the 3' terminus of AA-tRNA is analogous to the interaction of that terminus with EF-Tu and it is suggested that EF-Tu is specific for 2'-O-AA-tRNA.

Effect of nucleotide substitution on the peptidyltransferase activity of 2'(3')-O-(aminoacyl) oligonucleotides.

Seven 2'(3')-O-(aminoacyl) trinucleotides with structures derived from the 3'-terminal C-C-A sequence of aa-tRNA via nucleotide substitutions were investigated as acceptor substrates in the peptidyltransferase reaction and as inhibitors of substrate binding to the peptidyltransferase A site. It was found that all tested compounds were active in both systems, although substitution in the first and second nucleotide position results in some decrease of acceptor activity. Remarkably, replacement of natural cytidylic acid residues in C-C-A-Phe with guanylic acid moieties resulted only in a small decrease of acceptor or binding activity. The results indicate that the acceptor sequence of aa-tRNA is not probably engaged in base pairing with a sequence of 23S RNA during its interaction with the peptidyltransferase A site.

Enzymatic binding of aminoacyl transfer ribonucleic acid to ribosomes: the study of binding sites of 2' and 3' isomers of aminoacyl transfer ribonucleic acid.

The mechanism of enzymatic binding of AAtRNA to the acceptor site Escherichia coli ribosomes has been studied using the following aminoacyl oligonucleotides as models of the 3' terminus of AA-tRNA: C-A-Phe, C-A-(2'-Phe)H, and C-A(2'H)Phe. T-psi-C-Gp was used as a model of loop IV of tRNA. The EF-T dependent binding of Phe-tRNA to ribosomes (in the presence of either GTP or GMPPCP) and the GTPase activity associated with EF-T dependent binding of the Phe-tRNA were inhibited by C-A-Phe,C-A(2'Phe)H, and C-A(2'H)Phe. These aminoacyl oligonucleotides inhibit both the formation of ternary complex EF-Tu-GTP-AA-tRNA and the interaction of this complex with the ribosomal A site. The uncoupled EF-Tu dependent GTPase (in the absence of AA-tRNA) was also inhibited by C-A-Phe, C-A(2'Phe)H, and C-A(2'H)Phe, while nonenzymatic binding of Phe-tRNA to the ribosomal A site was inhibited by C-A-Phe and C-A(2'-Phe)H, but not by C-A(2'H)Phe. The tetranucleotide T-psi-C-Gp inhibited both enzyme binding of Phe-tRNA and EF-T dependent GTP hydrolysis. However, inhibition of the latter reaction occured at a lower concentration of T-psi-C-Gp suggesting a specific role of T-psi-C-Gp loop of AA-tRNA in the GTPase reaction. The role of the 2' and 3' isomers of AA-tRNA during enzymatic binding to ribosomes is discussed and it is suggested that 2' leads to 3' transacylation in AA-tRNA is a step which follows GTP hydrolysis but precedes peptide bond formation.

Aminoacyl-tRNA-elongation factor Tu-ribosome interaction leading to hydrolysis of guanosine 5'-triphosphate.

We investigated the elongation factor Tu (EF-Tu) dependent binding of Phe-tRNA and Phe-tRNAs with the nicks at positions 46, 37, and 17 to the Escherichia coli 70S ribosome-poly(U)-tRNAPhe complex. Binding of Phe-tRNA1-45 + 47-76, Phe-tRNA1-36 + 38-76, or Phe-tRNA1-16 + 17-76 to the 70S ribosome has been found to be poly(U) X tRNA dependent and, similar to that of intact Phe-tRNA, is inhibited by the antibiotic thiostrepton. We have further found that, contrary to a previous report [Modolell, J., Cabrer, B., Parmeggiani, A., & Vazquez, D. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 1796], the EF-Tu-ribosome GTPase mediated by Phe-tRNA is not inhibited by thiostrepton; rather, the drug stimulates the endogenous GTPase of the EF-Tu X 70S ribosome. Phe-tRNA fragments 47-76, 38-76, and 17-76 all promote the EF-Tu X GTPase reaction in the presence of 70S ribosome-poly(U)-tRNAPhe yeast. Moreover, since the GTPase-promoting activities of both the short and long fragments are similar, it appears that the most important aminoacyl transfer ribonucleic acid (aa-tRNA) interaction with EF-Tu occurs alongside its 3' quarter. Thiostrepton slightly stimulates the GTPase activity of these Phe-tRNA fragments. Although the Phe-tRNA1-36 + 38-76 cannot bind to poly(U) during its binding to 70S ribosomes, its binding at high Mg2+ concentration occurs at the A site. Thus, most of the bound modified Phe-tRNA functions as the acceptor in the peptidyltransferase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Recognition of the 3' terminus of 2'-O-aminoacyl transfer ribonucleic acid by the acceptor site of ribosomal peptidyltransferase.

The interaction of the 3' terminus of 2'- and 3'-O-aminoacyl-tRNA with the peptidyltransferase A site of Escherichia coli ribosomes has been studied using the following aminoacyl oligonucleotides as models of the 3' terminus of AA-tRNA: C-A-Phe, C-A(2'Phe)H, C-A(2'H)Phe, C-A(2'Phe)Me, C-A(2'Me)Phe, C-A(2'Gly)H, and C-A(2'H)Gly. The transfer of Ac-(14C)Phe from the Ac-(14C)Phe-tRNA-oly(U)-70S ribosome complex to puromycin (10-4 and 10-5 M) was inhibited by C-A-Phe, C-A(2'Phe)H, C-A(2'H)Phe, C-A(2'Gly)H, and C-A(2'H)Gly. Kinetic analysis of the inhibition of Ac(14C)Phe-puromycin formation by C-A(2'Phe)H failed to show simple competitive inhibition. Binding of C-A-C-C-A-(14C)Phe to 70S ribosomes in the presence of an excess of deacylated tRNA was also inhibited by C-A-Phe, C-A(2'Phe)H, C-A(2'H)Phe, C-A(2'Phe)Me, and C-A(2'Me)Phe. It appears that the acceptor site of peptidyltransferase can recognize the 3' terminus of either 2'- or 3'-O-AA-tRNA, with preference for the 2' isomer. It therefore follows that 2'-O-AA-tRNA amy be bound to ribosomes prior to peptide bone formation and that 3'-O-AA-tRNA, which is used exclusively by peptidyltransferase as an acceptor, is supplied by 2' leads to 3' transacylation occuring at the peptidyltransferase A site.

Inhibition of chloramphenicol binding to Escherichia coli 70S ribosomes by 2'(3')-O-aminoacyl-dinucleoside phosphates derived from the aminoacyl-tRNA acceptor terminus.

The effect of 2' and 3'-O-aminoacyl-dinucleoside phosphates cytidylyl(3'-5')-2'(3')-O-L-phenyl-alanyladenosine (I), cytidylyl(3'-5')-3'-deoxy-2'-O-L-phenylalanyladenosine (IIa), cytidylyl(3'-5')-2'-deoxy-3'-O-L-phenylalanyladenosine (IIIa), cytidylyl(3'-5')-3'-deoxy-2'-O-glycyladenosine (IIb), cytidylyl(3'-5')-2'-deoxy-3'-O-glycyladenosine (IIIb), cytidylyl(3'-5')-3'-deoxy-2'-O-L-leucyladenosine (IIc), cytidylyl(3'-5')-2'-deoxy-3'-O-L-leucyladenosine (IIIc), cytidylyl(3'-5')-3'-O-L-phenylalanyladenosine (IIId) as analogs of the 2'(3')-aminoacyl-tRNA termini, on chloramphenicol binding to 70S Excherichia coli ribosomes was investigated. The association constants (Kb) of the investigated compounds were determined by the equilibrium dialysis method. Based on the constancy of Kb over the range of inhibitor concentration, it was determined that the binding site of the 2' isomers IIa-IIc overlaps with the chloramphenicol site, whereas the variability of Kb for the 3' isomers IIIb, IIIc and especially IIIa seems to indicate that they do not achieve a complete fit. The consistently higher values of the Kb values for the 3' isomers IIIa-IIIc relative to that of the 2' isomers IIa-IIc also indicate a stabilization of the binding of the former due to a specific interaction between its amino acid portion and a ribosomal site.

Spectroscopic properties of various 2'(3')-O-aminoacyldinucleoside phosphates analogous to the 3' terminus of AA-tRNA.

Hypochromicity and circular dichroism data are reported for the 2' and 3'-0-aminiacyldinucleoside phosphates cytidylyl-(3'-5')-2'(3')-0-L-phenylalanyl-adenosine, cytidylyl-(3'-5')-2'-deoxy-3'-0-L-phenylalanyladenosine, cytidylyl-(3'-5')-2'-deoxy-3'-0-glycyladenosine, and cytidylyl-(3'-5')-3'-deoxy-2'-0-L-phenylalanyladenosine, all of which can act as analogs of the 3' terminus of AA-tRNA in various partial reactions of protein biosynthesis. Although all these systems have a 2'-OH group in the furanose of the 3'-residue, differences exist in the extent and/or mode of base-base overlap for most of them, except for cytidylyl-(3'-5')-2'(3')-0-L-phenylalanyladenosine and cytidylyl-(3'-5')-3'-deoxy-2'-0-L-phenylalanyladenosine. It is concluded that the biological activity of the above analogs is affected both by the position of the aminoacyl group and the stacking properties of the bases.