Template-free ribosomal synthesis of polypeptides from aminoacyl-tRNA. Polyphenylalanine synthesis from phenylalanyl-tRNALys.

Misacylated phenylalanyl-tRNALys, just as lysyl-tRNALys, but not phenylalanyl-tRNAPhe, have been shown to serve as substrates for ribosomal synthesis of polypeptides (polyphenylalanine and polylysine, respectively) in the absence of a template polynucleotide (poly(A)). The conclusion was made that it is the structure of tRNA that determines the ability of the aminoacyl-tRNALys to participate in peptide elongation on ribosomes without codon-anticodon interactions.

Template-independent synthesis of guanosine tetra- and pentaphosphates on ribosomes.

It is shown that Escherichia coli ribosomes carrying poly(Lys)-tRNA can form (p)ppGpp in the presence of stringent factor in the absence of the poly(A) template. Template-independent synthesis of (p)ppGpp is suppressed by tetracycline and partially decreases if deacylated tRNA is omitted.

Template-free ribosomal synthesis of polypeptides from aminoacyl-tRNAs.

In the present paper it has been demonstrated that Escherichia coli ribosomes in the absence of messenger polynucleotides are capable of synthesizing some polypeptides from aminoacyl-tRNAs as substrates. EF-Tu induced binding of aminoacyl-tRNA, ribosomal peptidyl transferase and EF-G-promoted translocation are strictly required for this template-free elongation. Typical ribosomal inhibitors such as tetracycline, chloramphenicol, phenylboric acid, fusidic acid have been shown to inhibit the template-free synthesis of polypeptides. The synthesis requires GTP cleavage; a non-cleavable analog of GTP, guanyl-5'-yl methylenediphosphonate does not maintain the synthesis. Among sixteen different aminoacyl-tRNAs studied as substrates for the ribosomal template-free synthesis of polypeptides Lys-tRNA, Ser-tRNA, Thr-tRNA and Asp-tRNA were the best. Gly-tRNA, Glu-tRNA, Val-tRNA, Arg-tRNA, Ala-tRNA and Leu-tRNA as substrates gave relatively low levels of the polypeptide synthesis on nonprogrammed ribosomes. Pro-tRNA, Phe-tRNA, Asn-tRNA, Met-tRNA, Ile-tRNA and Gln-tRNA were practically inactive as substrates for the template-free elongation. No correlation has been found between the abilities of the aminoacyl-tRNAs to serve as substrates for the template-free elongation and their activities in template-free binding to ribosomes.

[Translation of RNA 4 from plant brome mosaic virus in an Escherichia coli cell-free system with pure protein factors in translation]. / Transliatsiia RNK 4 rastitel'nogo virusa mozaiki kostra v beskletochnoi sisteme Escherichia coli s chistymi belkovymi faktorami transliatsii.

Translation by RNA 4 from plant brome mosaic virus coding for the virus coat protein in an E. coli cell-free system with pure factors of translation has been studied. It has been shown that the initiation of translation by this mRNA depends completely on the three E. coli initiation factors. Optimal ionic conditions for the formation of the initiatory 70S times fMet-tRNA times RNA 4 complex have been found. It has been shown that this complex is stable in conditions of zonal centrifugation. On the basis of reaction with puromycin it has been determined that the initiatory fMet-tRNA in this complex occupies the donor-tRNA-binding site of the ribosome. By the competence of the initiatory ribosomal complex for binding with Ser-tRNA (serine is the N-terminal amino acid in the virus coat protein) it can be concluded that the ribosomal and the E. coli initiation factors recognize the initiatory codon of the RNA r from brome mosaic virus. Peptide synthesis induced by RNA 4 has been obtained on E. coli ribosomes with five pure factors of translation: IF-1, IF-2A, IF-3, EF-Tu or (Tu--Ts) and EF-G. The dependence of elongation on the Mg2+ concentration in the medium at RNA 4 translation has been determined.

Ribosomal translocation assayed by the matrix-bound poly(uridylic acid) column technique.

The system of translation of cellulose-bound poly(uridylic acid) by Escherichia coli ribosomes has been used for preparation of pre-translocation state ribosomes in columns. Translocation has been induced by passing the elongation factor G (EF-G) with GTP or its non-cleavable analog (guanosine 5'-[beta, gamma-methylene]triphosphate) through the column. A method for quantitative comparison of translocation rates, and thus of effectiveness of translocation-inducing factors, has been proposed. The method is based on an analysis of the profile of deacylated tRNA elution resulting from translocation in the column. The determination of the rate and amount of translocation has been done under different ionic conditions. It has been found that the Mg2+ concentration is a decisive factor of translocation in vitro: at high Mg2+ (30 mM) EF-G cannot induce translocation, and lowering the Mg2+ concentration (to 10 mM) is required for EF-G to become effective. Sufficiently low Mg2+ (3 mM) itself has proved to induce fast and complete translocation, without EF-G.

[Sequence of events in the process of factor-promoted translocation in the ribosome]. / Posledovatel'nost' sobytii protsesse faktor-promotiruemoi translokatsii na ribosome.

A system of translation of matrix-bound poly(U) by purified Escherichia coli ribosomes was used to obtain pre-translocation state ribosomes in columns and then to induce translocation under controlled conditions by passing the elongation factor G (EF-G) with the non-cleavable GTP analog (guanylyl-methylene diphosphonate). It has been shown that translocation in the ribosome, checked by the release of deacylated tRNA, as well as by the puromycin reaction, is induced by the attachment of EF-G (with the non-cleavable GTP analog) to the ribosome and not by its detachment. In accordance with this, the ionic conditions under which the affinity of EF-G with the GTP analog to the ribosome is increased (NH4Cl instead of KCl, a lowered ionic strength) have been also found to be more effective for translocation. On the other hand, it has been shown that the detachment (removal) of EF-G is a strict pre-requisite for the appearance of competence to bind the next aminoacyl-tRNA, and thus for a continuation of the elongation cycle. A conclusion is made that the mechanical shifts of products and substrates, such as peptidyl-tRNA and deacylated tRNA, within the ribosome in the process of translocation are promoted only by the affinity of EF-G to the ribosome and does not depend on the cleavage of GTP. On the basis of the results obtained, the following sequence of events is deduced for the process of EF-G-promoted translocation: 1) interaction of EF-G.GTP with the pre-translocative ribosome, 2) translocation displacements of products and substrates, including the release of deacylated tRNA (probably conjugated with the shift of mRNA), 3) GTP hydrolysis, 4) release of EF-G and GTP from the post-translocated ribosome.

[Translocation in ribosomes induced by elongation factor G without cleavage of GTP. Study using a solid phase translation system in columns]. / Translokatsiia v ribosomakh, indutsiruemaia faktorom élongatsii G bez raspada GTP. Izuchenie pri pomoshchi tverdofaznoi sistemy transliatsii v kolonkakh.

Columns containing ribosomes translating poly(U) covalently bound with cellulose (solid-phase translating system) were used to study translocation in ribosomes. It is shown that the passing of elongation factor G (EF-G) with the non-cleavable analog of GTP (GMP-PCP) through a column containing pre-translocated ribosomes results in the increase of competence for puromycin (i. e. to the transition of pre-translocated peptidyl-tRNA into the post-translocated state) just as in the case of the passing of EF-G with GTP. On the other hand, it is shown that the passing of EF-G with GMP-PCP through a column with pre-translocated ribosomes makes them capable of binding the next aminoacyl-tRNA (i. e. leads to the vacation of the ribosomal A-site). Thus, by means of the two independent tests it is shown that EF-G-promoted translocation in the ribosome can proceed without GTP hydrolysis. On the basis of the data obtained, a controlled step-wise elongation of polypeptide with the participation of EF-G without GTP cleavage has been carried out in the solid-phase column system of translation.

A study of codon-dependent binding of aminoacyl-tRNA with the ribosomal 30-S subparticle of Escherichia coli. Determination of the active-particle fraction and binding constants in different media.

Titration of isolated Escherichia coli ribosomal 30-S particles with [14C]phenylalanyl-tRNA in the presence of poly(uridylic acid) was used for a quantitative assay of codon-dependent binding of aminoacyl-tRNA with the small ribosomal subparticle. The technique has allowed the estimation both of the fraction of "active" 30-S subparticles capable of forming the 30-S - poly(U) - phenylalanyl-tRNA complexes and the equilibrium constants of phenylalanyl-tRNA binding in different media. Heterogeneity of the ternary complexes formed has been revealed: at least two classes of complexes differing in stability have been observed. The stability of the 30-S - poly(U) - phenylalanyl-tRNA complexes has been shown to decrease with the lowering of the Mg2+ concentration, the increase of K+ concentration and the addition of urea. The stability of the complexes increases with the increase of Mg2+ concentration, with the addition of ethanol and decrease of temperature. It is demonstrated that the fraction of actively binding 30-S particles also varies in different medium conditions; it decreases with the increase of ionic strength (K+) and with the addition of urea, and increases with the increase of Mg2+ concentration and addition of ethanol.