Alexander S. Spirin

Folding of firefly luciferase during translation in a cell-free system.

In vitro synthesis of firefly luciferase and its folding into an enzymatically active conformation were studied in a wheat germ cell‐free translation system. A novel method is described by which the enzymatic activity of newly synthesized luciferase can be monitored continuously in the cell‐free system while this protein is being translated from its mRNA. It is shown that ribosome‐bound polypeptide chains have no detectable enzymatic activity, but that this activity appears within a few seconds after luciferase has been released from the ribosome. In contrast, the renaturation of denatured luciferase under identical conditions occurs with a half‐time of 14 min. These results support the cotranslational folding hypothesis which states that the nascent peptides start to attain their native tertiary structure during protein synthesis on the ribosome.

Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage

We have recently isolated and characterized a novel protein associated with Escherichia coli ribosomes and named protein Y (pY). Here we show that the ribosomes from bacterial cells growing at a normal physiological temperature contain no pY, whereas a temperature downshift results in the appearance of the protein in ribosomes. The protein also appears in the ribosomes of those cells that reached the stationary phase of growth at a physiological temperature. Our experiments with cell‐free translation systems demonstrate that the protein inhibits translation at the elongation stage by blocking the binding of aminoacyl‐tRNA to the ribosomal A site. The function of the protein in adaptation of cells to environmental stress is discussed.

Ribosomal Translocation: Facts and Models

Publisher Summary This chapter classifies the models concerning the different aspects of ribosomal translocation and discusses them in the light of todays factual grounds. It briefly reviews the two-tRNA-binding-sites (A and P) model for the ribosomal elongation cycle. Intermediate and additional tRNA binding positions may exist without detriment to the main scheme. A three-tRNA-binding-site model where the sequence of events differs from the two-tRNA-binding-sites (A and P) model is also mentioned in the chapter. The energetics models of ribosomal translocation are classified into two groups. One group includes the models where ribosomal ligands, in particular tRNAs, are actively pushed or pulled, mechanically or electrostatically, from one position to another at the expense of the energy of coupled GTP hydrolysis (GTP-driven translocation). The other group of models implies that the translocational displacements are thermodynamically spontaneous and proceed as ribosome-channeled diffusional movements of the ligands, the role of EF-G being considered as an entropic catalyst and GTP as a cleavable effector of EF-G (EF-G-catalyzed translocation). The preference is given to the second group of models. The kinematic and topographical models of ribosomal translocation are also discussed in the chapter.

Studies on the structure of ribosomes: II. Stepwise dissociation of protein from ribosomes by caesium chloride and the re-assembly of ribosome-like particles

The dissociation of ribosomal protein from Escherichia coli ribosomes under the influence of high CsCl concentrations was studied. Upon incubation of ribosomes in 5 M -CsCl containing 2 × 10−3 M -MgCl2, a definite portion of protein is dissociated from the ribosomes, turning normal 50 s and 30 s ribosomal subunits (37% protein) into protein-deficient 43 s and 28 S particles called A-particles (containing about 30% protein). Centrifugation of the analogous suspension in CsCl so as to remove dissociated protein from the equilibrium mixture by flotation leads to the formation of ribonucleoprotein particles even more deficient in protein (about 20% protein); at 10−3 M -MgCl2 they have sedimentation coefficients of So20, w = 28 s and 22 s and, in several of their characteristics, strongly resemble natural chloramphenicol CM-particles. It was shown that the dissociation of protein from ribosomal particles under the action of high CsCl concentrations seems to be reversible. The chlorampheni-col-like 28 S + 22 s particles easily re-associate with dissociated ribosomal protein in buffer solutions in the presence of magnesium ions, yielding 43 s + 28 s A-particles at 10−3 M -MgCl2 and 70 s + 50 s + 30 s ribosome-like particles at 10−2 M -MgCl2. The re-assembled ribosomal particles (70s, 50 s and 30 s) have the same protein content (37%) and the same buoyant density in CsCl equilibrium gradients (p = 1·64 g/cm3), as the original ribosomes. This in vitro reconstruction may be looked upon as a model of natural biogenesis (self-assembly) of bacterial ribosomes. The fact that both the dissociation of protein from ribosomes and their re-association are not gradual but proceed by discrete steps, without intervening stages, suggests the existence of groups of co-operatively organized protein molecules, that is, of ordered quaternary structures, within ribosomal particles.

Regulation of protein synthesis at the elongation stage New insights into the control of gene expression in eukaryotes

There are many reports which demonstrate that the rate of protein biosynthesis at the elongation stage is actively regulated in eukaryotic cells. Possible physiological roles for this type of regulation are: the coordination of translation of mRNA with different initiation rate constants; regulation of transition between different physiological states of a cell, such as transition between stages or the cell cycle; and in general, any situation where the maintenance of a particular physiological state is dependent on continuous protein synthesis. A number of covalent modifications of elongation factors offer potential mechanisms for such regulation. Among the various modifications of elongation factors, phosphorylation of eEF‐2 by the specific Ca2+/calmodulin‐dependent eEF‐2 kinase is the best studied and perhaps the most important mechanism for regulation of elongation rate. Since this phosphorylation is strictly Ca2+‐dependent, and makes eEF‐2 inactive in translation, this mechanism could explain how changes in the intracellular free Ca2+ concentration may regulate elongation rate. We also discuss some recent findings concerning elongation factors, such as the discovery of developmental stage‐specific elongation factors and the regulated binding of eEF‐1α to cytoskeletal elements. Together, these observations underline the importance of the elongation stage of translation in the regulation of the cellular processes essential for normal cell life.

Enzymatic activity of the ribosome-bound nascent polypeptide

Firefly luciferase was shown to be completely folded and thus enzymatically active immediately upon release from the ribosome [Kolb et al. (1994) EMBO J. 13, 3631–3637]. However, no luciferase activity was observed while full‐length luciferase was attached to the ribosome as a peptidyl‐tRNA, probably because the C‐terminal portion of the enzyme is masked by the ribosome and/or ribosome‐associated proteins. Here we have demonstrated that the ribosome‐bound enzyme acquires the enzymatic activity when its C‐terminus is extended by at least 26 additional amino acid residues. The results demonstrate that the acquisition of the final native conformation by a nascent protein does not need the release of the protein from the ribosome.

Gene expression in a cell-free system on the preparative scale

A cell-free system for preparative gene expression is described. It is composed of DNA-free Escherichia coli extract and added plasmid DNA; coupled transcription-translation proceeds with a continuous flow of the feeding solution containing nucleoside triphosphates and amino acids. The system works at a high constant rate for tens of hours. The yield of synthesised proteins after 20-50 h is hundreds of micrograms from 1 ml of the reaction mixture. Electrophoretic analysis of translation products confirms synthesis of proteins of the expected molecular mass.

Ribosome as a molecular machine

General principles of structure and function of the ribosome are surveyed, and the translating ribosome is regarded as a molecular conveying machine. Two coupled conveying processes, the passing of compact tRNA globules and the drawing of linear mRNA chain through intraribosomal channel, are considered driven by discrete acts of translocation during translation. Instead of mechanical transmission mechanisms and power‐stroke ‘motors’, thermal motion and chemically induced changes in affinities of ribosomal binding sites for their ligands (tRNAs, mRNA, elongation factors) are proposed to underlie all the directional movements within the ribosomal complex. The GTP‐dependent catalysis of conformational transitions by elongation factors during translation is also discussed.

The ribosome as a conveying thermal ratchet machine.

The research group of Professor Andrey Belozersky with whom I started my academic career in 1955 consisted of two parts: one was located at the Department of Plant Biochemistry, Moscow State University, and the other at the A. N. Bach Institute of Biochemistry, Academy of Sciences of the USSR. This biochemical group was one of the most creative in the country. It was world-renowned because of several important discoveries in the field of nucleic acid studies. In the thirties of the last century, it succeeded in settling the question of the universal occurrence of two known types of nucleic acids, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), in living matter. At that time, many biochemists believed that RNA is a characteristic component of plants and fungi, whereas DNA (designated as “thymonucleic acid” or “animal nucleic acid”) belongs to the animal kingdom. The presence of DNA in plant cells raised doubts, as the positive cytochemical Feulgen reaction in plant cell nuclei was the only indirect evidence. Belozersky and colleagues were the first to isolate thymine and then DNA (thymonucleic acid) from higher plants (1, 2), thus proving the universal occurrence of DNA. The next series of studies was carried out on bacteria (3) and demonstrated that both RNA and DNA were present there, again confirming the idea of the universality of the occurrence of both types of nucleic acids in organisms of different phylogenetic kingdoms. At the same time, the studies on bacteria showed that these organisms were deserving of special attention because of the high content of nucleic acids in their cells. During the years from 1939 to 1947, the systematic studies of the content of nucleic acids in bacteria of various taxonomic families, of different ages, and under different physiological conditions were performed in both subgroups headed by Belozersky (4). The high level of nucleic acids in cells was postulated to be in direct relation to their biological activities, growth rate, and cell proliferation.

Does the channel for nascent peptide exist inside the ribosome? Immune electron microscopy study.

MS2 phage RNA‐directed synthesis of an N‐terminal polypeptide of the phage coat protein on Escherichia coli 70 S ribosomes was initiated in a cell‐free system with the N‐dinitrophenyl derivative of methionyl‐tRNAMet F and performed in the absence of tyrosine, lysine, cysteine and methionine. As a result, the translating ribosomes carried peptides up to 42 amino acid residues in length with the dinitrophenyl hapten at the N‐ends. Using the immune electron microscopy technique the positions of the nascent peptide N‐ends on the 70 S ribosomes have been visualized. It has been found that (i) the N‐ends of nascent peptides of these lengths are accessible to antibodies, (ii) the exit site of a nascent peptide is the pocket between the base of the central protuberance and the L1 ridge on the 50 S subunit, i.e. presumably its peptidyl transferase center, and (iii) the further pathway of a nascent peptide seems to proceed along the groove on the external surface of the 50 S subunit.