RESUMO
The shape of non-fractionated Escherichia coli transfer RNA molecules in solution was investigated using small-angle X-ray scattering during the years 1960-1962 at the Centre de Recherche sur les Macromolécules in Strasbourg. The innermost region of the scattering curve yielded the average molecular weight (Mr) and the radius of gyration (Rg) of the particles, whereas the experimental data at large angles could be approximated at best by the scattering curve of a kinked rod-shaped molecule. The simplest model that was compatible with Mr, Rg, and the mass per unit length of the rod was a boomerang-shaped particle made of two double helical stems connected by a sharp kink. This model that eventually proved similar to the high-resolution L-shaped structure, was presented in my Ph.D. dissertation (J. Witz, Etude de la structure de quelques polynucléotides en solution par diffusion centrale des rayons X, Ph.D. dissertation, University of Strasbourg, France, 1964) but has never been published in detail. It is the purpose of this note to recall this story.
Assuntos
Conformação de Ácido Nucleico , RNA de Transferência/química , RNA de Transferência/história , Difração de Raios X/história , Escherichia coli/genética , História do Século XX , Editoração/história , Espalhamento de Radiação , Raios XRESUMO
In this article, I review how our research on RNA began, how it led us to demonstrate the single-stranded nature of RNA, and the ways in which it differs from double-stranded DNA. It was based on the development of a method for the isolation of undegraded rRNA and the observation that in rRNA preparations due to their viscosity behavior resemble a flexible, contractile coil. In support of this assumption, birefringence of flow measurements showed that rRNA solutions gave moderate positive values, which disappeared upon addition of salt. This is in contrast with DNA solutions where considerable negative birefringence persists even in the presence of salt. Further studies on RNA showed a close correlation of the ionic strength dependencies of optical rotation, optical density and hydrodynamic properties. These early results indicated that rRNA and tRNA possess a significant secondary structure. I then review the basis of the hairpin model for the secondary structure of RNA and finally, summarize current understanding of the tertiary structure of RNA.
Assuntos
Conformação de Ácido Nucleico , RNA/história , História do Século XX , Israel , Concentração Osmolar , RNA/química , RNA/isolamento & purificação , RNA Bacteriano/química , RNA Bacteriano/história , RNA Bacteriano/isolamento & purificação , RNA Ribossômico/química , RNA Ribossômico/história , RNA Ribossômico/isolamento & purificação , RNA de Transferência/química , RNA de Transferência/história , RNA Viral/química , RNA Viral/história , ViscosidadeRESUMO
This review highlights studies by Lev L. Kisselev and his colleagues on the initial and terminal stages of protein biosynthesis, which cover the period of the last 45 years (1961-2006). They investigated spatial structure of tRNAs, structure and functions of aminoacyl-tRNA-synthetases of higher organisms, and the final step of protein synthesis, termination of translation. L. Kisselev and his team have made three major contributions to these fields of molecular biology; (i) they proposed the hypothesis on the role of anticodon triplet of tRNA in recognition by cognate aminoacyl-tRNA synthetase, which has been experimentally confirmed and is now included in textbooks; (ii) identified primary structures and functions of two eukaryotic protein factors (eRF1 and eRF3) playing a pivotal role in translation termination; (iii) characterized a structural basis for stop codon recognition by eRF1 within the ribosome and discovered the negative structural elements of eRF1, limiting its recognition of one or two stop-codons.
Assuntos
Biossíntese de Proteínas , Aminoacil-tRNA Sintetases/história , Aminoacil-tRNA Sintetases/metabolismo , História do Século XX , História do Século XXI , Iniciação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/história , Fatores de Terminação de Peptídeos/metabolismo , RNA de Transferência/genética , RNA de Transferência/história , RNA de Transferência/metabolismoRESUMO
In 1946, (14)C-cyanide made its appearance as an offshoot of the Atomic Energy Program. Our colleague Robert Loftfield built it into (14)C-alanine by the Strecker synthesis, and a lusty program directed toward uncovering the unknown mechanism of protein synthesis grew out of this beginning. The necessity for an undiscovered series of steps and enzymes was soon evident. A cell free system was developed, and a succession of components necessary for this new pathway tumbled out. ATP dependence, amino acid activation, the ribosome as the site of polypeptide formation, discovery of tRNA as the translation molecule linking the gene and protein sequence, and GTP as the essential energy ingredient in peptide chain extension all appeared from our laboratory within the next decade. A little later the AP(4)N family, whose functions remain imperfectly defined, of intracellular molecules was discovered. Isolation of specific species of RNA became a high priority, and we sequenced a small segment of the 3' end of the Rous sarcoma virus, just inside the poly(A) tail, at the same time the Gilbert group at Harvard was sequencing the 5' end. The sequence identity and polarity of the two ends suggested a circular intermediate in replication and predicted correctly that a synthetic antisense oligonucleotide targeted against this sequence might be a specific inhibitor of replication. More recently, we have evolved a technique that appears to achieve a trinucleotide insertion into tissue culture cells bearing a specific Delta508 mRNA triplet deletion, resulting in phenotypic reversion in the tissue culture.