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V A Erdmann

Publications and source records attributed to V A Erdmann.

At least 73 records · Page 4Linked to original sources
PubMed1994

The dynamic conformation of plant cytoplasmic 5S rRNAs.

Recently we have proposed a new three-dimensional model of plant 5S rRNAs structure. To verify this proposal we present here new data on RNase T1 digestion and hydroxyl radical hydrolysis of lupin and wheat germ 5S rRNAs at various buffer and temperature conditions. Interestingly, the guanosine residues 85-87 in the loop D of these RNAs, are resistant to RNase T1 at native but not at denaturating conditions. On the other hand, the reaction of 5S rRNA with the hydroxyl radicals showed different reactivity of many nucleotides in various parts of the molecule and suggest conformational changes, which occur mostly in the loops. The experimental data clearly support involvement of the nucleotides occupying conserved positions in the loops in the tertiary interactions in plant 5S rRNA structure.

Base Sequence↗
PubMed1994

A-Z-RNA conformational changes effected by high pressure.

This paper reports evidence obtained by circular dichroism (CD) spectroscopy measurements indicating that two oligoribonucleotide duplexes with the alternating purine-pyrimidine sequences r(GC)6 or r(AU)6 change their A-RNA conformation under high pressure. Under the high-pressure conditions at which B-Z-DNA transition easily occurs, RNA acquires a conformation which only differs slightly from that of A-RNA. However, exposure of r(GC)6 or r(AU)6 to high pressure (6 kbar) in the presence of 5 M NaCl causes a conformation change of both oligoribonucleotide duplexes from their A- to their Z-RNA form. The departure of RNA or DNA duplexes from their original conformations under high pressure depends on the water structure itself and involves displacing an active (structural) water molecule outside the nucleic acid molecules. Experiments carried out until now in many laboratories have shown that B-Z or A-Z transitions of DNA or RNA, respectively, do not depend on the conditions applied, but the common mechanism for these processes seems to be dehydration. This same effect can be observed either at high salt concentrations or in the presence of an alcohol or at high pressure. Our results also support the view that the higher stability of RNA compared with DNA duplexes is due to the strong interaction of the 2'-hydroxyl groups of RNA with water molecules.

Circular Dichroism↗
PubMed1994

A new type of RNA editing. 5S ribosomal DNA transcripts are edited to mature 5S rRNA.

The primary structure of the 5S rRNA from Arabidopsis thaliana was determined. A comparison of this nucleotide sequence with that of 5S rRNA gene showed two differences. Furthermore, we compared all plant 5S rRNA and 5S rDNA sequences known to date and found that lack of colinearity is widespread among higher plant 5S rRNAs and occurred mainly in the double stranded regions. To explain this, we suggest a mechanism which converts putative products of pseudogene ("cryptogene") to mature 5S rRNA molecule. This kind of editing mechanism functions primarily in stems of 5S rRNA in order, to correct mispairing and thereby restoring the Watson-Crick base pairs. This idea could explain why so many different 5S rRNA genes or gene like sequences and only one 5S rRNA species exist in the plant cell. The editing can serve as a new mechanism of regulation of 5S rRNA synthesis in addition to transcription of 5S rRNA gene.

Base Sequence↗
PubMed1993

Sequence of the gene encoding ribosomal protein L11 from Thermus thermophilus HB8.

The complete nucleotide sequence of the gene encoding ribosomal protein L11 from the extreme thermophilic eubacterium, Thermus thermophilus HB8, was determined. L11 amino acid (aa) sequences from mesophilic and halophilic organisms, as well as from another thermophiles, were compared with the T. thermophilus L11 aa sequence.

Amino Acid Sequence↗
PubMed1993

Role of the D arm and the anticodon arm in tRNA recognition by eubacterial and eukaryotic RNase P enzymes.

Truncated precursor tRNAs lacking the D arm or anticodon arm were studied in vitro as substrates for RNase P enzymes from Escherichia coli, Thermus thermophilus (eubacteria), and HeLa. Deletion of the D arm still allowed 5'-processing by E. coli RNase P, but strongly impaired maturation by T. thermophilus and HeLa extracts. In contrast, deletion of the anticodon arm had no influence on processing by RNase P activities from all three organisms. Inhibition kinetics and gel retardation studies showed that deletion of the D arm leads to low-affinity binding to E. coli RNase P RNA (M1 RNA). However, the E. coli enzyme appears to form sufficiently strong contacts in the region of the T arm, acceptor stem, and CCA terminus to still allow productive enzyme-substrate interaction even in the absence of the structural contribution provided by the D arm. Pb(2+)-induced hydrolysis of a tRNAGly from T. thermophilus gave identical cleavage patterns in the D arm and anticodon loop in the absence and presence of E. coli M1 RNA, whereas lead hydrolysis was strongly reduced at the CUCCAA 3'-terminus due to the presence of the enzyme.

Anticodon↗
PubMed1993

Gel retardation analysis of E. coli M1 RNA-tRNA complexes.

We have analyzed complexes between tRNA and E. coli M1 RNA by electrophoresis in non-denaturing polyacrylamide gels. The RNA subunit of E. coli RNase P formed a specific complex with mature tRNA molecules. A derivative of the tRNA(Gly), endowed with the intron of yeast tRNA(ile) (60 nt), was employed to improve separation of complexed and unbound M1 RNA. Binding assays with tRNA(Gly) and intron-tRNA(Gly) as well as analysis of intron-tRNA/M1 RNA complexes on denaturing gels showed that one tRNA is bound per molecule of M1 RNA. A tRNA carrying a truncation as small as the 5'-nucleotide had a strongly reduced affinity to M1 RNA and was also a weak competitor in the cleavage reaction, suggesting that nucleotide +1 is a major determinant of tRNA recognition and that the thermodynamically stable tRNA-M1 RNA complex is relevant for enzyme function. Binding was shown to be dependent on the M1 RNA concentration in a cooperative fashion. Only a fraction of M1 RNAs (50-60%) readily formed a complex with intron-tRNA(Gly), indicating that distinct conformational subpopulations of M1 RNA may exist. Formation of the M1 RNA-tRNA(Gly), complex was very similar at 100 mM Mg++ and Ca++, corroborating earlier data that Ca++ is competent in promoting M1 RNA folding and tRNA binding. Determination of apparent equilibrium constants (app Kd) for tRNA(Gly) as a function of the Mg++ concentration supports an uptake of at least two additional Mg++ ions upon complex formation. At 20-30 mM Mg++, highest cleavage rates but strongly reduced complex formation were observed. This indicates that tight binding of the tRNA to the catalytic RNA at higher magnesium concentrations retards product release and therefore substrate turnover.

Base Sequence↗
PubMed1993

Prokaryotic elongation factor Tu is phosphorylated in vivo.

Covalent modification of proteins by phosphate transfer reactions constitutes a major mechanism of regulation in higher eukaryotes. Recently, phosphorylation of eukaryotic elongation factors has been described. Analysis of Escherichia coli proteins revealed several of them to be phosphorylated. Various lines of evidence lead us to conclude that one of these proteins is identical to elongation factor (EF) Tu, which can be phosphorylated in vivo at one of its threonine residues. Structural analysis showed that one fragment of the phosphorylated EF-Tu is highly resistant to tryptic digestion. Phosphorylation of eubacterial EF-Tu is not restricted to the E. coli factor but could also be demonstrated for Thermus thermophilus HB8 EF-Tu. Overexpression of tufA did not increase the number of EF-Tu molecules to be phosphorylated. This may indicate that a constant but limited amount of EF-Tu is modified, possibly for a specific function. Phosphorylation of EF-Tu could also be demonstrated in vitro. Upon analysis of subcellular fractions the highest kinase activity was found in the ribosomal fraction of E. coli. Protein sequencing of both the in vivo and in vitro phosphorylated protein revealed position 382 as the modified threonine residue.

Amino Acid Sequence↗
PubMed1992

Cleavage efficiencies of model substrates for ribonuclease P from Escherichia coli and Thermus thermophilus.

We compared cleavage efficiencies of mono-molecular and bipartite model RNAs as substrates for RNase P RNAs (M1 RNAs) and holoenzymes from E. coli and Thermus thermophilus, an extreme thermophilic eubacterium. Acceptor stem and T arm of pre-tRNA substrates are essential recognition elements for both enzymes. Impairing coaxial stacking of acceptor and T stems and omitting the T loop led to reduced cleavage efficiencies. Small model substrates were less efficiently cleaved by M1 RNA and RNase P from T. thermophilus than by the corresponding E. coli activities. Competition kinetics and gel retardation studies showed that truncated tRNA substrates are less tightly bound by RNase P and M1 RNA from both bacteria. Our data further indicate that (pre-)tRNA interacts stronger with E. coli than T. thermophilus M1 RNA. Thus, low cleavage efficiencies of truncated model substrates by T. thermophilus RNase P or M1 RNA could be explained by a critical loss of important contact points between enzyme and substrate. In addition, acceptor stem--T arm substrates, composed of two synthetic RNA fragments, have been designed to mimic internal cleavage of any target RNA molecule available for base pairing.

Base Sequence↗
PubMed1992

Biochemical and NMR spectroscopy evidence for a new tertiary A-U base pair in lupin ribosomal 5 S RNA structure.

The new model for the tertiary structure of ribosomal 5 S rRNA from plants recently proposed by some of us has been already supported by RNase H digestions in the presence of complementary oligodeoxynucleotides. These results are confirmed now by the new biochemical and NMR spectroscopy data. Diethylpyrocarbonate (DEP) and monoperphthalic acid (MPA) are the reagents with the high specificity toward single-stranded adenosine residues. Our experiments clearly show that under native conditions adenosine 100 (A100) of lupin 5 S rRNA is not available for reaction toward these reagents. However under denaturing conditions this residue reacts with DEP and MPA. The detailed analysis of the lupin 5 S rRNA by NMR spectra provide the data on the specific interaction of A100-U53. Thus, we have seen for the first time the NMR signal due to the A100-U53 tertiary base pair, which as we believe, stabilizes interactions between loops B and E.

Adenine↗
PubMed1992

Sequence of the tufA gene encoding elongation factor EF-Tu from Thermus aquaticus and overproduction of the protein in Escherichia coli.

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB-encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182-191). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coli is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coli using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays.

Amino Acid Sequence↗
PubMed1992

Structural analysis of three prokaryotic 5S rRNA species and selected 5S rRNA--ribosomal-protein complexes by means of Pb(II)-induced hydrolysis.

Lead ions have been applied to the structural analysis of 5S rRNA from Thermus thermophilus, Bacillus stearothermophilus and Escherichia coli. Based on the distribution of Pb(II)-induced cleavages, some minor modifications of the consensus secondary structure model of 5S rRNA are proposed. They include the possible base pairing between nucleotides at positions 11 and 109, as well as changes in secondary interactions within the helix B region. The 'prokaryotic arm' region is completely resistant to hydrolysis in the three RNA species, suggesting that it is a relatively stable, highly ordered structure. Hydrolysis of E. coli 5S rRNA complexed with ribosomal protein L18 shows, besides the shielding effect of the bound protein, a highly enhanced cleavage between A108 and A109. It supports the concept that the major L18-induced conformational change involves the junction of helices A, B and D.

Autoradiography↗
PubMed1992

Different conformational forms of Escherichia coli and rat liver 5S rRNA revealed by Pb(II)-induced hydrolysis.

Different stable forms of Escherichia coli and rat liver 5S rRNA have been probed by Pb(II)-induced hydrolysis. In the native A forms of 5S rRNA, Pb2+ reveal single-stranded RNA stretches and regions of increased conformational flexibility or distorted by the presence of bulged nucleotides. Hydrolysis of urea/EDTA-treated E. coli 5S rRNA (B form) shows the presence of two strong helical domains; helix A retained from the A form and a helix composed of RNA regions G33-C42 and G79-C88. Other RNA regions resistant to hydrolysis may be involved in alternative base pairing, causing conformational heterogeneity of that form. Pb(II)-induced hydrolysis distinguishes two different forms of rat liver 5S rRNA; the native A form and the form obtained by renaturation of 5S rRNA in the presence of EDTA. Pb(II)-hydrolysis data suggest that both forms are highly structured. In the latter form, the orientation of the bulged C66 is changed with respect to helix B. At the same time, a new helical segment is possibly formed, composed of nucleotides from helix C and loop c on one side and from helix E and loop d' on the other.

Animals↗
PubMed1992

Unfolding of the tertiary structure of specific tRNA and ribosomal 5S RNA from plants as studied with hydroxyl radicals.

Ribosomal 5S RNA is present in all eubacterial and eukaryotic ribosomes. Despite a large amount of experimental data on the primary and secondary structures of these types of molecules, details of their tertiary structure and their precise function in protein biosynthesis are still not known. Recently we have proposed a new model for the tertiary structure of plant 5S rRNA. In this study we applied the Fe(II)-mediated cleavage reaction to test the model. The data presented here provide experimental evidence that in the 5S rRNA molecule only a few nucleotides are buried in the tertiary structure. Similar experiments performed with methionine initiator tRNA gave results which imply the difference in its structure when compared with the X-ray structure of yeast tRNAPhe.

Base Composition↗
PubMed1992

Identification of the gene encoding transcription factor NusG of Thermus thermophilus.

The nusG gene of Thermus thermophilus HB8 was cloned and sequenced. It is located 388 bp downstream from tufB, which is followed by the genes for ribosomal proteins L11 and L1. No equivalent to secE preceding nusG, as in Escherichia coli, could be detected. The nusG gene product was overproduced in E. coli. A rabbit antiserum raised against the purified recombinant NusG reacted exclusively with one protein band of T. thermophilus crude extracts in Western blot (immunoblot) analyses, and no cross-reaction of the antiserum with E. coli NusG was observed. Recombinant NusG and the reacting T. thermophilus wild-type protein had identical sizes on sodium dodecyl sulfate-polyacrylamide gels. T. thermophilus and E. coli NusG have 45% identical and 22.5% similar amino acids, and similarities between the two proteins are most pronounced in carboxy-terminal regions. The T. thermophilus nusG gene could not rescue a nusG-deficient E. coli mutant strain.

Amino Acid Sequence↗
PubMed1992

Dynamic structure of transfer RNA in solution monitored by reaction with hydroxyl radicals.

The dynamic structure of initiator and elongator tRNAs was analyzed using a very sensitive reaction with hydroxyl radicals. The main target for this reagent is the ribose moieties not buried (accessible) in the tertiary structure of the RNA molecule. At variable time, temperature and magnesium concentrations, some nucleoside residues of lupin initiator tRNA or yeast tRNA(Phe) become accessible or not depending on the actual tRNA conformation. The nucleotides in the thymidine stem of yeast tRNA(Phe) and amino acid stem in both tRNAs do not change their reactivity and conformation. Also the reactivities of the nucleosides of the anticodons are not changing. Our data clearly suggest that hydroxyl radicals can be very useful for the analysis of the tertiary structure of tRNA.

Autoradiography↗
PubMed1991

Analysis of the gene encoding the RNA subunit of ribonuclease P from T. thermophilus HB8.

The gene for the RNA subunit of ribonuclease P from the extreme thermophilic eubacterium T. thermophilus HB8 was cloned using oligonucleotide probes complementary to conserved regions of RNase P RNA subunits from proteobacteria. The monocistronic gene and its flanking regions were sequenced. The gene is enclosed by a promoter and a rho-independent terminator. Nuclease S1 protection analyses showed that the primary transcript is identical with the mature RNA, i.e. no processing events are involved. The stem and loop structure of the terminator remains part of the mature molecule. In vitro transcription of the cloned gene with purified RNA polymerase from T. thermophilus yields the same RNA product as in vivo, indicating that no other components except RNA polymerase are involved in the synthesis of the RNA. RNase P RNA from T. thermophilus cleaved a pre-tRNA(Tyr) from E. coli with highest efficiency between 55 degrees C and 65 degrees C. The T. thermophilus RNA, which has a G-C content of 86% in helical regions, displays several structural idiosyncrasies, although its secondary structure is similar to that of proteobacteria. Numerous invariable nucleotides in the structural core of eubacterial RNase P RNAs are also conserved in the RNA from the extreme thermophilic eubacterium.

Base Sequence↗
PubMed1991

Identification of the phosphorylation sites of the murine small heat shock protein hsp25.

Native phosphorylated mouse small heat shock protein hsp25 from Ehrlich ascites tumor cells was isolated and the in vivo phosphorylation sites of the protein were determined. Furthermore, native hsp25 was phosphorylated by the endogenous kinase(s) in a cell-free system as well as recombinant hsp25 was phosphorylated in vitro by protein kinase C and catalytic subunit of cAMP-dependent protein kinase. The two major phosphorylation sites of native and recombinant hsp25 were determined as Ser-15 and Ser-86. There are no differences in the hsp25 phosphorylation sites phosphorylated by the protein kinase C, the catalytic subunit of cAMP-dependent protein kinase and the unknown intracellular kinase(s). The serine residues identified exist in all known small mammalian stress proteins and are located in the conserved kinase recognition sequence Arg-X-X-Ser.

Amino Acid Sequence↗