Biomedical subjects

A Spirin

Publications and source records attributed to A Spirin.

5 recordsLinked to original sources

PubMed1994

Coupled replication-translation of amplifiable messenger RNA. A cell-free protein synthesis system that mimics viral infection.

Amplifiable messenger RNAs (Wu, Y., Zhang, D. Y., and Kramer, F. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11769-11773) were used as templates in coupled replication-translation reactions. These amplifiable mRNAs contained a preselected messenger sequence embedded within the sequence of MDV-1 RNA, which is a small, naturally occurring template for Q beta replicase. When these recombinant mRNAs were incubated in vitro in reactions that contained both an Escherichia coli cell-free translation system and Q beta replicase, the encoded protein was synthesized more efficiently than in corresponding reactions that did not contain Q beta replicase. Moreover, when coupled replication-translation reactions were carried out in a continuous-flow format (Spirin, A. S., Baranov, V. I., Ryabova, L. A., Ovodov, S. Yu., and Alakhov, Yu. B. (1988) Science 242, 1162-1164), the synthesis of biologically active protein continued for a prolonged period. The results suggest that the mechanism of replication and translation in coupled reactions is similar to the mechanism by which Q beta phage genomic RNA is simultaneously replicated and translated in Q beta-infected E. coli: protein synthesis occurs on nascent RNA strands; many more sense strands are synthesized than antisense strands; and the integrity of the messenger sequence is preserved because a relatively small number of antisense strands serve as master templates for the synthesis of new messenger strands.

Cell-Free System↗

PubMed1992

How are tRNAs and mRNA arranged in the ribosome? An attempt to correlate the stereochemistry of the tRNA-mRNA interaction with constraints imposed by the ribosomal topography.

Two tRNA molecules at the ribosomal A- and P-sites, with a relatively small angle between the planes of the L-shaped molecules, can be arranged in two mutually exclusive orientations. In one (the 'R'-configuration), the T-loop of the A-site tRNA faces the D-loop of the P-site tRNA, whereas in the other (the 'S'-configuration) the D-loop of the A-site tRNA faces the T-loop of the P-site tRNA. A number of stereochemical arguments, based on the crystal structure of 'free' tRNA, favour the R-configuration. In the ribosome, the CCA-ends of the tRNA molecules are 'fixed' at the base of the central protuberance (the peptidyl transferase centre) of the 50S subunit, and the anticodon loops lie in the neck region (the decoding site) of the 30S subunit. The translocation step is essentially a rotational movement of the tRNA from the A- to the P-site, and there is convincing evidence that the A-site must be located nearest to the L7/L12 protuberance of the 50S subunit. The mRNA in the two codon-anticodon duplexes lies on the 'inside' of the 'elbows' of the tRNA molecules (in both the S-type and R-type configurations), and runs up between the two molecules from the A- to the P-site in the 3' to 5'-direction. These considerations have the consequence that in the S-configuration the mRNA in the codon-anticodon duplexes is directed towards the 50S subunit, whereas in the R-configuration it is directed towards the 30S subunit. The results of site-directed cross-linking experiments, in particular cross-links to mRNA at positions within or very close to the codons interacting with A- or P-site tRNA, favour the latter situation. This conclusion is in direct contradiction to other current models for the arrangement of mRNA and tRNA on the ribosome.

Cross-Linking Reagents↗

PubMed1992

Structural dynamics of translating ribosomes.

We describe three groups of small angle neutron scattering (SANS) experiments with translating ribosomes: 1) regular protonated (normal abundance hydrogen) particles; 2) two isotopic hybrid particles which are reconstituted from one protonated and the other deuterated subunit; 3) four isotypic hybrid particles differing from each other by the extent of protein and RNA deuteration. Using the SANS contrast variation method the radii of gyration of protein and RNA components in both ribosomal subunits as well as the intersubunit distance in the pre- and post-translocation states were determined. The results obtained suggest the following model of the ribosome as a dynamic machine. The ribosome oscillates between two major conformers differing in geometrical dimensions. The 'active' (pulsating) part of the ribosome is the 30S subunit. We believe that the movement of its 'head' relative to the passive 50S subunit is the main mechanical act of translocation. The radius of gyration of the 30S subunit and the intersubunit distance change upon the movement. This is corroborated by neutron scattering data.

Bacterial Proteins↗

PubMed1991

Formation and crystallization of Thermus thermophilus 70S ribosome/tRNA complexes.

70S ribosomes from Thermus thermophilus are able to form ternary complexes with N-AcPhe-tRNAPhe from either Thermus thermophilus or Escherichia coli, in the presence of a short oligo(U) of six or nine uridines. A complex of N-AcPhe-tRNAPhe/(U)9/70S ribosome from Th. thermophilus was crystallized under the same conditions used for the growth of crystals from isolated ribosomes (S.D. Trakhanov, et al., (1987) FEBS Lett. 220, 319-322).

Crystallography↗

PubMed1987

Structural dynamic aspects of protein synthesis on ribosomes.

Three types of conformational changes in the translating ribosome are considered: (1) intersubunit movement (ribosome unlocking) during translocation; (2) L7/L12 stalk mobility affected by elongation factors; (3) change of tRNA residue during its transition from the A-site to the P-site. Relevant experimental data are reviewed.

Escherichia coli↗