Structure of the elongating ribosome: Arrangement of the two tRNAs before and after translocation

Abstract

The ribosome uses tRNAs to translate the genetic information into the amino acid sequence of proteins. The mass ratio of a tRNA to the ribosome is in the order of 1:100; because of this unfavorable value it was not possible until now to determine the location of tRNAs within the ribosome by neutron-scattering techniques. However, the new technique of proton-spin contrast-variation improves the signal-to-noise ratio by more than one order of magnitude, thus enabling the direct determination of protonated tRNAs within a deuterated ribosome for the first time. Here we analyze a pair of ribosomal complexes being either in the preor post-translocational states that represent the main states of the elongating ribosome. Both complexes were derived from one preparation. The orientation of both tRNAs within the ribosome and their mutual arrangement are determined by using an electron microscopy model for the Escherichia coli ribosome and the tRNA structure. The mass center of gravity of the (tRNA) 2 mRNA complex moves within the ribosome by 12 ؎ 4 Å in the course of translocation as previously reported. The main results of the present analysis are that the mutual arrangement of the two tRNAs does not change on translocation and that the angle between them is, depending on the model used, 110°؎ 10°before and after translocation. The translocational movement of the constant tRNA complex within the ribosome can be described as a displacement toward the head of the 30S subunit combined with a rotational movement by about 18°.The central machinery of the translational apparatus is the ribosome, which is one of the most complicated cellular structures. It separates into two subunits and consists of more than 50 components. The central RNA ligands of the ribosome during protein synthesis are tRNAs and mRNA. mRNA brings the genetic information to the ribosome where tRNAs are used to interpret the codon sequence of the mRNA in terms of an amino acid sequence in the growing peptide (for a recent synopsis see ref. 1). Data derived from cross-linking studies, chemical protection, and fluorescence measurements have led to the proposal of conflicting models for the tRNA positions in the elongating ribosome (2-4) because of uncertainties in the spatial assignment of ribosomal components. Thus, additional information on the location of tRNAs by using direct physical methods is necessary to evaluate and refine the models. Positions of tRNAs within the ribosome deduced from electron microscopy (EM) have been proposed recently (5, 6). Neutron-scattering analysis has been applied for direct localization of the mass center of gravity for the (tRNA) 2 mRNA complex present on the ribosome during elongation (7).Here we describe the relative arrangement of the tRNAs before and after translocation as derived from the neutronscattering data. The mutual arrangement of the tRNAs does not change on translocation from their pre-to their posttranslocational positions, and the angle between them remains constant at 110°Ϯ 10°. MATERIALS AND METHO...

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

“…Transfer RNAs (tRNAs) are the substrates used by ribosomes for synthesizing proteins+ To understand the mechanism of translation at the molecular level, it is essential to understand how tRNAs interact with the ribosome (Rheinberger et al+, 1981;Grajevskaja et al+, 1982;Kirillov et al+, 1983;Lill et al+, 1984)+ The anticodon arm of P-site tRNA interacts with mRNA in the vicinity of the cleft formed by the platform and the head of the 30S subunit (Lake, 1980;Gornicki et al+, 1984)+ The acceptor arms of A-site and P-site tRNAs interact with the peptidyl transferase center, which lies close to the base of the central protuberance of the 50S subunit (Ofengand, 1980;Olson et al+, 1982;Wower et al+, 1989)+ During peptide bond formation, the CCA-39 termini of A-site and P-site tRNAs must be proximal to allow peptidyl transfer, while their anticodons interact with adjacent codons on mRNA (Fairclough & Cantor, 1979a;Matzke et al+, 1980)+ Several models have been proposed for the arrangement of A-site and P-site tRNAs in the ribosome based on stereochemical considerations (Fuller & Hodgson, 1967;Woese, 1970;Rich, 1974;Sundaralingam et al+, 1975;Lake, 1977;Spirin & Lim, 1986;McDonald & Rein, 1987;Prabahakaran & Harvey, 1989;Nagano et al+, 1991;Easterwood et al+, 1994), fluorescence resonance energy transfer (FRET) (Johnson et al+, 1982;Paulsen et al+, 1983), crosslinking studies (Ofengand, 1980, Ofengand et al+, 1981Wower et al+, 1989Wower et al+, ,hotra et al+, 1998, and other methods (Spirin, 1983;Smith & Yarus, 1989;Wagenknecht et al+, 1989;Nierhaus et al+, 1998)+ Most of these models can be assigned to either the R (Rich, 1974) or S (Sundaralingam et al+, 1975) orientations+ In the R orientation, the T loop of A-site tRNA ...…”

Protection Patterns of tRNAs Do Not Change during Ribosomal Translocation

“…On the other hand, it is known that the mRNA moves three nucleotides through the ribosome in the course of translocation (22), and also the mass centers of gravity of both the mRNA (23) and the two tRNAs move 12 Å within the ribosome (24), in good agreement with the length of one codon. Most important, the two tRNAs turn by an angle of at least 20°d uring translocation (25,26). Such a turn means that the elbows of the two L-shaped tRNAs change their positions considerably and move even more than 12 Å during translocation.…”

Neutrons for biologists: a beginner's guide, or why you should consider using neutrons

“…Deuterated lipids make the system even more flexible such that individual lipid species can be observed separately, for example during phase separation (Pencer et al 2005). Finally, nucleic acids, which are matched at 65 per cent D 2 O, can be seen separately from proteins as in a study of tRNAs in the deuterated ribosome (Nierhaus et al 1998). …”