Biomedical subjects

J Wadzack

Publications and source records attributed to J Wadzack.

8 recordsLinked to original sources

PubMed1998

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

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 pre- or 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)2mRNA complex moves within the ribosome by 12 +/- 4 A 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 degrees +/- 10 degrees 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 degrees.

Binding Sites↗

PubMed1998

Small angle scattering in ribosomal structure research: localization of the messenger RNA within ribosomal elongation states.

Besides EM and biochemical studies small angle scattering (SAS) examinations have contributed significantly to our current knowledge about the ribosomal structure. SAS does not only allow the validation of competing models but permits independent model building. However, the major contribution of SAS to ribosomal structure research derived from its ability to reveal the spatial distribution of the individual ribosomal components (57 in the E. coli ribosome) within the ribosomal structure. More recently, an improved scattering method (proton-spin contrast variation) made it possible also to address the question of mapping functional ligands in defined ribosomal elongation states. Here, we review the contributions of SAS to the current understanding of the ribosome. Furthermore we present the direct localization of a small mRNA fragment within 70S elongation complexes and describe its movement upon the translocation reaction. The successful mapping of this fragment comprising only about 0.6% of the total mass of the complex proves that proton-spin contrast-variation is a powerful tool in modern ribosome research.

Peptide Chain Elongation, Translational↗

PubMed1997

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

A new technique for neutron scattering, the proton-spin contrast-variation, improves the signal-to-noise ratio more than one order of magnitude as compared to conventional techniques. The improved signal enables small RNA ligands within a large deuterated ribonucleic acid-protein complex to be measured. We used this technique to determine the positions of the two tRNAs within the elongating ribosome before and after translocation. Using a four-sphere model for each of the L-shaped tRNAs, unequivocal solutions were found for the localization of the mass centre of both tRNAs. The centre of gravity is located in the interface cavity separating the ribosomal subunits near the neck of the 30 S subunit. It moves during translocation by 12(+/-4) A towards the head of the 30 S subunit and slightly towards the L1 protuberance of the 50 S subunit.

Base Sequence↗

PubMed1996

In vivo deuteration of transfer RNAs: overexpression and large-scale purification of deuterated specific tRNAs.

Structural investigations of tRNA complexes using NMR or neutron scattering often require deuterated specific tRNAs. Those tRNAs are needed in large quantities and in highly purified and biologically active form. Fully deuterated tRNAs can be prepared from cells grown in deuterated minimal medium, but tRNA content under this conditions is low, due to regulation of tRNA biosynthesis in response to the slow growth of cells. Here we describe the large-scale preparation of two deuterated tRNA species, namely D-tRNAPhe and D-tRNAfMet (the method is also applicable for other tRNAs). Using overexpression constructs, the yield of specific deuterated tRNAs is improved by a factor of two to ten, depending on the tRNA and growth condition tested. The tRNAs are purified using a combination of classical chromatography on an anion exchange DEAE column with reversed phase preparative HPLC. Purification yields nearly homogenous deuterated tRNAs with a chargeability of 1400-1500 pmol amino acid/A260 unit. The deuterated tRNAs are of excellent biological activity.

Chromatography, High Pressure Liquid↗

PubMed1995

Self-coded 3'-extension of run-off transcripts produces aberrant products during in vitro transcription with T7 RNA polymerase.

More than 70% of the RNA synthesized by T7 RNA polymerase during run-off transcription in vitro can be incorrect products, up to twice as long as the expected transcripts. Transcriptions with model templates indicate that false transcription is mainly observed when the correct product cannot form stable secondary structures at the 3'-end. Therefore, the following hypothesis is tested: after leaving the DNA template, the polymerase can bind a transcript to the template site and the 3'-end of the transcript to the product site and extend it, if the 3'-end is not part of a stable secondary structure. Indeed, incubation of purified transcripts with the polymerase in transcription conditions triggers a 3'-end prolongation of the RNA. When two RNAs of different lengths are added to the transcription mix, both generate distinct and specific patterns of prolonged RNA products without any interference, demonstrating the self-coding nature of the prolongation process. Furthermore, sequencing of the high molecular weight transcripts demonstrates that their 5'-ends are precisely defined in sequence, whereas the 3'-ends contain size-variable extensions which show complementarity to the correct transcript. Surprisingly, a reduction of the UTP concentration to 0.2-1.0 mM in the presence of 3.5-4.0 mM of the other NTPs leads to faithful transcription and good yields, irrespective of the nucleotide composition of the template.

Bacteriophage T7↗

PubMed1994

How the ribosome moves along the mRNA during protein synthesis.

The movement of a ribosome along the mRNA was assessed by the following experimental strategy. mRNAs were synthesized which contained a short coding sequence with at least four codons and a 32P label at one end and an oligo(C) sequence at the other end. When these mRNAs were fixed on the ribosome with tRNAs specific for the defined codons, the oligo(C) stretches were partially outside of the ribosome, whereas the labeled ends were inside the ribosome and thus protected. The overhanging oligo(C) regions were trimmed with the cytidyl-specific RNase CL3 identifying the nucleotides of the mRNAs, which emerged from the ribosome. An mRNA enters the ribosome at nucleotide 18 +/- 1, when counting starts at the first nucleotide of the P-site codon, and leaves the ribosome at nucleotide -21 +/- 2. The ribosome does not move at either side upon A-site occupation but does so at both sides simultaneously upon translocation. The results further indicate that most of the mRNA stretches downstream and upstream of the coding region are not required for the translocation reaction. It is therefore likely that the tRNAs are pulling the mRNA through the ribosome via codon-anticodon interactions in the course of translocation.

Amino Acid Sequence↗

PubMed1993

Large-scale preparation of fully deuterated cell components. Ribosomes from Escherichia coli with high biological activity.

Some applications of NMR and of neutron scattering require fully deuterated biological material which should be highly active and available in large quantities. These requirements are hardly compatible since full deuteration is achieved easily only if cells are grown in minimal media. This condition used in standard batch fermentation results in both low yields and reduced activities of the biological mass. Here we report a method which combines the apparently incompatible requirements taking advantage of a recent observation according to which the appearance of growth inhibiting extracellular products could be prevented. The method was applied for growing Escherichia coli cells, strain MRE600rif (resistance against high doses of rifampicin is used as selection marker) on partially deuterated media (76% and 84% D2O) with glucose as carbon source and on deuterated acetate and succinate with 100% D2O when full deuteration was to be achieved. The essential point for preserving the log-phase character of the cells is that the cultivation is carried out at substrate limiting conditions thus keeping the growth rate at low levels (for glucose the growth rate, mu < or = 0.35 h-1, for acetate/succinate mu < or = 0.1 h-1) which avoids the accumulation of the substrate or of by-products in the medium. Our data suggest that acetate is a main extracellular component for accompanying or triggering the transition from logarithmic growth to stationary phase of E. coli cells cultivated on glucose as carbon source. The cells were first grown in fed-batch to high cell densities (above 50 g wet cells/l) under conditions of substrate limitations. A steady-flow fermentation followed keeping the growth rate at about mu of 0.1 h-1. Cells were harvested in kg quantities, the extracted ribosomes showed a normal complement of proteins, contained intact rRNA and were fully active. The ribosomal protein and rRNA fractions could be efficiently reconstituted to highly active particles. In the case of full deuteration a matching point of 120% (tentative D2O scale) was achieved. The reported method facilitates the preparation of deuterated biological material for applications in NMR and neutron scattering analysis.

Acetates↗

PubMedDate not supplied

The elongating ribosome: structural and functional aspects.

We determined the positions and arrangements of RNA ligands within the ribosome with a new neutron-scattering technique, the proton-spin contrast-variation. Two tRNAs were bound to the ribosome in the pre-translocational and the post-translocational state. The mass centre of gravity of both tRNAs resides at the subunit interface of the body of the 30S subunit. Both tRNAs are separated by an angle of 50-55 degrees, and their mutual arrangement does not change during translocation. The mass centre of gravity moves by 13 +/- 3 A (1A = 0.1 nm) during translocation, corresponding well with the length of one codon. Using an RNase-digestion technique, the length of the mRNA sequence covered by the ribosome was determined to be 39 +/- 3 nucleotides before and after translocation. The ribosome moves like a rigid frame along the mRNA during translocation. In contrast, both tRNAs seem to be located on a movable ribosomal domain, which carries the tRNAs before, during, and after translocation, leaving the microtopography of the tRNAs with the ribosome unaltered. This conclusion was derived from an analysis of the contract patterns of thioated tRNAs on the ribosome. The results have led to a new model of the elongation cycle, which reinterprets the features of the previous "allosteric three-sites model" in a surprisingly simple fashion. Finally, a mutational analysis has identified a single nucleotide of the 23S rRNA essential for the peptidyltransferase activity.

Amino Acid Sequence↗