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Biomedical subjects

V A Erdmann

Publications and source records attributed to V A Erdmann.

At least 127 records · Page 7Linked to original sources
PubMed1987

Structural analysis of 5S rRNA, 5S rRNA-protein complexes and ribosomes employing RNase H and d(GTTCGG).

The hybridization of d(GTTCGG) to eubacterial 5S rRNAs, 5S rRNA-protein complexes, 70S ribosomes and 50S and 30S ribosomal subunits was investigated. This oligonucleotide, which may be considered to be an analogue of the T psi CG loop of tRNAs, was chosen in order to investigate a possible interaction between tRNAs with ribosomal components during protein synthesis. The hybridization was analysed by RNase H hydrolysis studies and, in the case of the ribosomes and ribosomal subunits, in addition with the radioactively labelled oligodeoxyribonucleotide in binding studies. The results obtained lead to the conclusion that nucleotides in loop c, i.e. positions 42-47, are available for oligonucleotide interaction in free Escherichia coli and Bacillus stearothermophilus 5S rRNAs and not available in the corresponding 5S rRNA-protein complexes. The 70S ribosomes and ribosomal subunits did not interact with the oligonucleotide. Under the assumption that d(GTTCGG) is an analogue of the T psi CG loop of tRNAs and in view of the results obtained, we conclude that in the unprogrammed ribosomes the T psi CG loop of tRNAs does not interact via standard Watson-Crick base pairs with the ribosomal 5S, 16S or 23S RNAs.

Bacillus↗
PubMed1987

An unusual rRNA operon constellation: in Thermus thermophilus HB8 the 23S/5S rRNA operon is a separate entity from the 16S rRNA operon.

We succeeded in identifying a promoter element within 200 base pairs upstream a transcriptional unit comprising only a 23S rRNA, 5S rRNA and a tRNA(gly) gene in Thermus thermophilus HB8 [1, 2]. This element shows a high degree of homology to the -35 and -10 consensus sequences for promoters described for Escherichia coli [3, 4]. The promoter activity was measured by the induction of the synthesis of functional chloramphenicol acetyltransferase in Escherichia coli. A region located at the transcriptional start, rich in guanosines and cytidines, is very similar in sequence to the one believed to be under stringent control in stable RNA and ribosomal protein genes of Escherichia coli [5]. Employing nuclease S1 protection we were able to determine the in vivo start of transcription, which was identical with the in vitro start using Escherichia coli RNA-polymerase. Furthermore we identified sequences in the region following the origin of transcription, which are homologous to sections in Escherichia coli rrn promoter-leader regions responsible for antitermination. Our finding of a promoter immediately preceding a 23S/5S rRNA operon proves a transcriptional decoupling of the 16S rRNA genes, a situation so far unprecedented among prokaryotes.

Acetyltransferases↗
PubMed1987

Evolution of organisms and organelles as studied by comparative computer and biochemical analyses of ribosomal 5S RNA structure.

The results documented in this publication demonstrate that for evolutionary studies the ribosomal 5S rRNA is a suitable object for such an investigation and that as many methods as possible should be consulted. In this study the results of biochemical and chemical experiments were combined with those of computer sequence analyses, and they revealed that these methods complement each other nicely. We are currently at a state at which we are able to well define the secondary structures of the 5S rRNAs for eubacteria, organelles, archaebacteria, and eukaryotes and we are even able to propose a secondary structure for a Ur-5S rRNA. It is also clear that in the future the present studies should be continued and extended in such a way that the tertiary structures of these molecules will become known.

Animals↗
PubMed1986

Analysis of the RNA structural elements involved in the binding of the transcription factor III A from Xenopus laevis.

Xenopus laevis 5S rRNA isolated from 7S particles or transcribed in vitro is found to adopt two alternative conformations. These two conformers contain different structural elements within the major TF III A binding domain, which, when isolated as RNA fragments, still interact with the transcription factor. Chemical modification of easily accessible adenines in their N-1 position does not have any measurable effect on the binding of 5S rRNA to TF III A. These observations are in support of the idea that only a small amount of conserved sequence information is required for the binding of the transcription factor, whereas specific secondary structure features seem to be essential.

Animals↗
PubMed1986

Small cytoplasmic RNAs from human placental free mRNPs. Structure and their effect on in vitro protein synthesis.

A new family of small cytoplasmic RNA species (scRNAs) was found to be associated with human term-placental free messenger ribonucleoprotein particles (mRNPs). Placental scRNAs strongly inhibit translation of both homologous and heterologous mRNAs in a cell-free rabbit reticulocyte system. scRNAs could be resolved into at least four different RNA species. One of the RNA molecules, scRNA species 1, was the most potent protein synthesis inhibitor found among the placental scRNAs. The nucleotide sequence of the scRNA species 1 was determined. In spite of its short length, scRNA species 1 still exhibited a very strong inhibitory effect on the in vitro protein synthesis. scRNAs were found to be complexed with proteins in the form of scRNPs. Proteins of these complexes enhanced the inhibitory effect of scRNAs on in vitro translation. Experiments provided evidence that inhibition of in vitro protein synthesis by the scRNAs is not dependent upon mRNA concentration. However, inhibition can be overcome by increasing the ratio lysate/scRNAs, thus suggesting that scRNAs act on some essential component of the cell-free system. The degree of inhibition is decreased when scRNAs are added after the start of translation, suggesting that scRNAs (or scRNPs) interfere with the initiation stage of translation, probably acting on an initiation factor(s). Placental scRNAs are unique in their size, being smaller than other known scRNAs. Their association with free cytoplasmic repressed mRNPs in human placenta suggests that scRNAs play a role in the regulation of mRNP metabolism and, consequently, in the control of mRNA translation.

Base Sequence↗
PubMed1986

Genomic organization of rDNA in Pseudomonas aeruginosa.

We have examined the number and organization of rRNA genes in Pseudomonas aeruginosa by hybridization of restriction nuclease digests of genomic DNA to 3'-32P-labelled 23 S, 16 S and 5 S rRNAs and corresponding labelled DNA from the rrnB operon of Escherichia coli. The immediate conclusion from these hybridization data is that there are 4 transcriptional units coding for rDNA in P. aeruginosa. We report here a putative model of the genomic organization of all 4 rDNA operons.

Chromosome Mapping↗
PubMed1986

Improved procedure for the isolation of a double-strand-specific ribonuclease and its application to structural analysis of various 5S rRNAs and tRNAs.

An improved method for the isolation of a double-strand-specific RNase from snake venom is presented. This RNase, called CSV, was used to cleave yeast tRNAPhe and tRNA2Glu and tRNAfMet from Escherichia coli. In addition these RNAs and E. coli tRNAPhe were examined with the single-strand-specific nuclease S1. The results are discussed in terms of the specificity of CSV RNase and the structure of tRNAs. S1 nuclease digestions at increasing temperatures allowed the melting of tertiary and secondary structure to be monitored. 5S rRNA from E. coli, Thermoplasma acidophilum and the chloroplasts of Spinacia oleracea were digested with CSV and S1. The information these results give on the secondary-structural differences between different classes of 5S rRNA are discussed. Supporting evidence is found for tertiary interactions between hairpin loop c and internal loop d of eubacterial 5S rRNA.

Animals↗
PubMed1985

Functional domains of the Xenopus laevis 5S gene promoter.

To study the fine structure of the Xenopus laevis somatic 5S gene internal control region, we have created 15 different transversions using mutagenic oligonucleotide primers. The effects of these mutations on 5S DNA transcription in vitro as well as on stable complex formation with transcription factor TF III A and TF III C in crude nuclear extracts were analyzed. Mutations in the common class III 5' promoter element (nucleotides 50-61 in the 5S gene) interfere with transcription activity and stable complex formation whenever they contradict the tDNA box A consensus sequence. The second promoter element is defined by a major sequence block (nucleotides 80-89, box C) and two additional internal residues (70 and 71) at a distance of roughly one helical turn from both the major 3' and 5' control sequences; these two 3' elements contain the primary TF III A binding domain. The remaining nucleotides (62-69 and 71-79) when mutated do not interfere with transcription activity or factor binding and thus they constitute two spacer elements within a symmetrically structured 5S gene promoter. An increase in the relative spacing of box A and box C by insertion of 3 bp between nucleotides 66 and 67 leads to a drastic reduction in transcription activity and the ability to form a stable complex with TF III A and/or TF III C. Thus, accurate spacing is essential for the proper orientation of TF III A on 5S DNA and/or TF III C binding.

Animals↗
PubMed1985

Expression of plasmid encoded Escherichia coli 5S ribosomal ribonucleic acid in Pseudomonas putida.

The recombinant plasmid pNRK 36, which represents the plasmids RSF 1010, a small multicopy plasmid of the incompatibility group IncQ that confers resistance to streptomycin and sulfoamide to its host cells, and pKK 223-3, which contains the try-lac (tac) promoter followed by a polylinker and a DNA segment containing the 5S rRNA (rrn B) with the ribosomal RNA transcription terminators, was employed to transform Pseudomonas putida 2440 cells. The plasmid encoded 5 S rRNA from Escherichia coli was transcribed and processed properly in P. putida cells thus demonstrating for the first time the expression of a plasmid encoded ribosomal RNA in a heterologous system. The fact that the E. coli 5 S rRNA was not incorporated into assembled ribosomes suggests that the in vivo incorporation of 5 S rRNA into the 50 S ribosomal subunit is closely linked to 23 S rRNA and/or ribosomal protein synthesis.

Centrifugation, Density Gradient↗
PubMed1985

Point mutational analysis of the Xenopus laevis 5S gene promoter.

We have introduced C to T transitions into GC and CG base pairs of the Xenopus laevis somatic 5S gene coding region and its 5' flank in order to analyse their effects on transcription activity and regulation. These studies allow us to differentiate between the two promoter elements and their spacer, within the internal control region. Mutations within the 5' element which dissent from the corresponding tRNA consensus sequence reduce transcription activity substantially without significantly affecting transcription factor (TF) III A binding. Mutations in the spacer region have no pronounced effect on transcription. The 3' promoter element is found to extend to position 97, since mutations in this region interfere with transcription activity. This may be, at least partially, attributable to a reduced competition strength for TF III A.

Animals↗
PubMed1985

RNA structural dynamics: pre-melting and melting transitions in E. coli 5S rRNA.

The temperature dependent transition from duplex to a single strand in E. coli 5S ribosomal RNA is a multistep process, and it involves intermediate states. We have analyzed these structural dynamics by chemical modification of cytidines and by single strand specific nuclease digestions. This combined approach led to the characterization of premelting and melting transitions within individual structural segments of the native macromolecule, which we feel may find general application to the structure of biological polyribonucleotides: 1) G-C base pairs at the termini of helices are relatively unstable and they readily undergo premelting transition. 2) Internal G-U/A-U rich stretches of helices exhibit dynamic premelting properties. 3) Hairpin loops have a relatively stronger destabilizing effect than internal loops. 4) Bulge loops destabilize the neighbouring base pairs. 5) Melting of helical segments occurs starting from the destabilizing structures listed above, preferentially from the helix termini. E. coli 5S rRNA has been shown to adopt different conformations. The presence of urea leads to induction of enhancement in the sensitivity for nuclease S1 at several nucleotide positions. The possibility of structural rearrangements will be discussed.

Base Sequence↗
PubMed1984

Structural requirements for the interaction of 5S rRNA with the eukaryotic transcription factor IIIA.

In order to study the binding of the eukaryotic transcription factor IIIA to heterologous 5S rRNAs with a low degree of overall sequence conservation (less than 20%) we have utilized a transcription competition assay involving eubacterial, archaebacterial and eukaryotic 5S rRNAs. All the molecules inhibit Xenopus 5S rRNA transcription specifically, which suggests that only a small amount of specific conserved RNA sequences, if indeed any, are essential for the interaction of the transcription factor with the 5S rRNA molecule, whereas universal 5S rRNA secondary structure elements seem to be required. A fragment of Xenopus laevis oocyte 5S rRNA (nucleotides 41-120), which partially maintains the original 5S rRNA structure, also competes for TF III A. In vitro transcription of a naturally occurring mutant of the Xenopus laevis oocyte 5S rRNA gene, the pseudogene, which carries several point mutations within the TF III A binding domain is equally inhibited by exogenous Xenopus 5S rRNA.

Animals↗
PubMed1984

Comparative structural analysis of eubacterial 5S rRNA by oxidation of adenines in the N-1 position.

Adenines in free 5S rRNA from Escherichia coli, Bacillus stearothermophilus and Thermus thermophilus have been oxidized at their N-1 position using monoperphthalic acid. The determination of the number of adenine 1-N-oxides was on the basis of UV spectroscopic data of the intact molecule. Identification of the most readily accessible nucleotides by sequencing gel analysis reveals that they are located in conserved positions within loops, exposed hairpin loops and single-base bulge loops. Implications for the structure and function of 5S rRNA will be discussed on the basis of this comparative analysis.

Adenine↗