Direct localization of the tRNAs within the elongating ribosome by means of neutron scattering (proton-spin contrast-variation)

Wadzack, Jörg; Burkhardt, Nils; Jünemann, Ralf; Diedrich, Gundo; Nierhaus, Knud H.; Frank, Joachim; Penczek, Pawel A.; Meerwinck, W.; Schmitt, Matthias; Willumeit‐Römer, Regine; Stuhrmann, H. B.

doi:10.1006/jmbi.1996.0788

Cited by 35 publications

(18 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…For stabilization of the spin alignment the sample is kept at temperatures below 0.2 K. Both elongation states were measured two times for 1 week each. The functional integrity of the particles is not affected by this treatment as shown previously (7,9).…”

Section: Preparation Of Ribosomal Elongation Complexessupporting

confidence: 54%

“…7 contains a detailed description of sources, biochemical preparations, and the isolation and characterization of the ribosomal elongation complexes used as samples for neutronscattering analysis as well as for the preparation of the samples for proton-spin contrast variation. The respective complexes contained the RNA ligands (mRNA and two tRNAs) protonated in a fully deuterated ribosomal matrix in D 2 O milieu.…”

Section: Preparation Of Ribosomal Elongation Complexesmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…By polarization of the spins the signal from protonated components can be selectively enhanced. The proton spin alignment in the sample reaching about 70% was achieved by dynamic nuclear spin polarization (7,10). For stabilization of the spin alignment the sample is kept at temperatures below 0.2 K. Both elongation states were measured two times for 1 week each.…”

mentioning

confidence: 99%

See 3 more Smart Citations

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

Nierhaus

Wadzack

Burkhardt

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Section: Preparation Of Ribosomal Elongation Complexessupporting

confidence: 54%

Section: Preparation Of Ribosomal Elongation Complexesmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Nierhaus

Wadzack

Burkhardt

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…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).…”

Section: Contact Patterns Of Trnas Do Not Change During Translocationmentioning

confidence: 90%

Protection Patterns of tRNAs Do Not Change during Ribosomal Translocation

Dabrowski

Spahn

Schäfer

et al. 1998

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The translocation reaction of two tRNAs on the ribosome during elongation of the nascent peptide chain is one of the most puzzling reactions of protein biosynthesis. We show here that the ribosomal contact patterns of the two tRNAs at A and P sites, although strikingly different from each other, hardly change during the translocation reaction to the P and E sites, respectively. The results imply that the ribosomal micro-environment of the tRNAs remains the same before and after translocation and thus suggest that a movable ribosomal domain exists that tightly binds two tRNAs and carries them together with the mRNA during the translocation reaction from the A-P region to the P-E region. These findings lead to a new explanation for the translocation reaction.Ribosomes contain three tRNA binding sites, the A, P, and E site, viz. the A site where the decoding takes place, the P site, where the peptidyl-tRNA is located before peptide bond formation, and the E site, which is specific for deacylated tRNA (1-6). During elongation of the nascent peptide chain, each tRNA passes through the ribosomal binding sites in the sequence A 3 P 3 E. To elongate the nascent peptide chain by one amino acid, the ribosome goes through a cycle of reactions, the socalled elongation cycle. The three basic reactions of an elongation cycle are 1) occupation of the A site by an aminoacyl-tRNA according to the corresponding codon at the A site, 2) peptidebond formation, which transfers the already synthesized peptidyl residue to the aminoacyl-tRNA so that the resulting peptidyl-tRNA, prolonged by an amino acid, now resides at the A site, and 3) the translocation reaction, which moves the peptidyl-tRNA to the P site and the deacylated tRNA to the E site.Cross-linking and footprinting studies have identified components of the ribosome that interact with the tRNAs in the specific sites (reviewed in Refs. 7 and 8). Recently, a technique developed by Eckstein and co-workers (9) has been used to investigate the interactions of tRNAs with ribosomal binding sites (10). This method exploits the fact that the addition of iodine (I 2 ) causes a breakage of the sugar-phosphate backbone of phosphorothioated RNA (here tRNA). The cleavage works equally well with phosphates in single or double strands. However, if tight contacts of a distinct phosphate group of a thioated tRNA with a synthetase or a ribosomal component prevents the access of iodine, the phosphate group under observation is protected, and a cleavage is not observed at this position. Highly differentiated and distinct cleavage patterns were found for A and P site-bound tRNAs in the pre-translocational (PRE) 1 state (10). Here we show that the protection patterns of both tRNAs at the A and P sites in the PRE state hardly change when translocated to P and E sites, respectively. Because probably most of the protection patterns are caused by interactions of the tRNA with the respective ribosomal binding sites, the data suggest the existence of a movable ribosomal domain that binds tightly the two ...

show abstract

Contrast Variation

Trewhella¹

2002

Encyclopedia of Molecular Biology

View full text Add to dashboard Cite

Direct localization of the tRNAs within the elongating ribosome by means of neutron scattering (proton-spin contrast-variation)

Cited by 35 publications

References 35 publications

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

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

Protection Patterns of tRNAs Do Not Change during Ribosomal Translocation

Contrast Variation

Contact Info

Product

Resources

About