Biomedical subjects

R K Hartmann

Publications and source records attributed to R K Hartmann.

At least 37 records · Page 2Linked to original sources

PubMed1996

Mutational analysis of the joining regions flanking helix P18 in E. coli RNase P RNA.

We have studied variants of Escherichia coli RNase P RNA with base exchanges in the joining regions flanking helix P18, which form part of the ribozyme core structure. Mutant RNase P RNAs were analyzed for: (1) specific tRNA binding by gel retardation; (2) catalytic performance in single turnover reactions; (3) structural perturbations utilizing Pb2+ -induced hydrolysis; and (4) in vivo function by complementation analysis in E. coli RNase P mutant strains. Our in vitro experiments revealed that the base moieties of nucleotides (nt) 303 and 331 to 333 neither significantly contribute to tRNA binding or structural stabilization of RNase P RNA nor to active site chemistry. Single base exchanges at nt 300, 301, and 330 reduced tRNA binding, while having little effect on the catalytic rate, which demonstrates that these nucleotides are involved in forming the high affinity (pre-)tRNA binding site. In contrast, point mutations at the strictly conserved positions nt 328, 329, 334 and 335 reduced tRNA binding affinity as well as the catalytic rate, suggesting that these mutations additionally disrupted important interactions in the catalytic center. Probing by Pb2+ revealed that particularly the mutations that affected catalytic function most strongly perturbed a more extended region (nt 248 to 335) known to be involved in tRNA binding. Under high salt conditions (> or = 0.8 M NH4+), catalytic defects of the mutant RNase P RNAs were much less pronounced, suggesting that structural perturbations leading to increased electrostatic repulsion between phosphate groups were the main cause for observed functional defects. Only mutant C334 retained a largely increased pre-steady-state K(m(pss)) under high salt conditions. We conclude that the base at position 334 is directly involved in a contact crucial to pre-tRNA binding. A complementation analysis demonstrated the important role in vivo of the joining regions flanking helix P18. None of the bases could be mutated without affecting bacterial viability.

Base Composition↗

PubMed1996

Rp-deoxy-phosphorothioate modification interference experiments identify 2'-OH groups in RNase P RNA that are crucial to tRNA binding.

Ribose 2'-hydroxyls make a key contribution to the enormous structural and functional potential of RNA molecules. Here, we report the identification of 2'-deoxy modifications in the catalytic RNA subunit of RNase P from Escherichia coli that interfere with tRNA binding. This was accomplished by modification interference employing pools of RNase P RNA that carried a low level of Rp-deoxy-phosphorothioate (Rp-deoxyNMPalpha(S) ) modifications randomly distributed over its 380 nt. A gel retardation assay allowed us to separate RNase P RNA pools into tRNA-binding and nonbinding fractions. Differences in the intensity of phosphorothioate-specific iodine hydrolysis patterns of the two RNA fractions revealed positions where the Rp-deoxyNMPalpha(S) modification interferes with tRNA binding. A comparison with interference patterns obtained for the Rp-NMPalpha(S) modification alone has identified some 20 positions in the backbone of E. coli RNase P RNA where the functional defect caused by the Rp-deoxyNMPalpha(S) double modification is attributable to the 2'-deoxy modification (or possibly the C5 methyl group in the case of U residues because we used deoxyTMPalpha(S) for partial substitution of UMP). Most of the corresponding 2'-OH functions were localized in regions that have been reported to crosslink to photoreactive tRNA derivatives, suggesting that these 2'-hydroxyls are located along the tRNA binding interface of E. coli RNase P RNA. Our results indicate that the modification interference approach applied here will be useful generally to identify structurally and functionally important 2'-hydroxyls in large RNAs and ribozymes.

Animals↗

PubMed1995

Direct linkage of str-, S10- and spc-related gene clusters in Thermus thermophilus HB8, and sequences of ribosomal proteins L4 and S10.

We have analyzed the genomic region harboring str-S10-spc-related gene clusters in the thermophilic bacterium, Thermus thermophilus (Tt) HB8. This study was initiated for the purpose of isolating the gene encoding ribosomal (r-) protein S10 which is assumed to be involved in the antitermination of transcription at the rRNA-encoding genes in Bacteria. The S10-related gene cluster encodes the same set of r-proteins as in Escherichia coli. However, the gene coding for elongation factor Tu (the last gene of the str operon in E. coli) is separated by only eight nucleotides (nt) from the gene encoding r-protein S10 (the first gene of the S10 operon in E. coli), and the genes encoding r-protein S17 (the last gene of the S10 operon in E. coli) and L14 (the first gene of the spc operon in E. coli) overlap. This suggests that all three gene clusters are cotranscribed from a single promoter preceding the str-related operon. In addition, we determined the complete nt sequences of the Tt genes encoding r-proteins L4 and S10. Tt L4 shows the lowest degree of conservation among the known L4 r-proteins from Bacteria. Tt S10 has the highest proportion of basic amino acids (aa) and the lowest number of acidic aa, as compared with its homologues from Bacteria and Archaea, which might be related to its possible role in binding to the boxA RNA of nascent rRNA transcripts at high temperatures.

Amino Acid Sequence↗

PubMed1995

Precursor of C4 antisense RNA of bacteriophages P1 and P7 is a substrate for RNase P of Escherichia coli.

The C4 repressor of the temperate bacteriophages P1 and P7 inhibits antirepressor (Ant) synthesis and is essential for establishment and maintenance of lysogeny. C4 is an antisense RNA acting on a target, Ant mRNA, which is transcribed from the same promoter. The antisense-target RNA interaction requires processing of C4 RNA from a precursor RNA. Here we show that 5' maturation of C4 RNA in vivo depends on RNase P. In vitro, Escherichia coli RNase P and its catalytic RNA subunit (M1 RNA) can generate the mature 5' end of C4 RNA from P1 by a single endonucleolytic cut, whereas RNase P from the E. coli rnpA49 mutant, carrying a missense mutation in the RNase P protein subunit, is defective in the 5' maturation of C4 RNA. Primer extension analysis of RNA transcribed in vivo from a plasmid carrying the P1 c4 gene revealed that 5'-mature C4 RNA was the predominant species in rnpA+ bacteria, whereas virtually no mature C4 RNA was found in the temperature-sensitive rnpA49 strain at the restrictive temperature. Instead, C4 RNA molecules carrying up to five extra nucleotides beyond the 5' end accumulated. The same phenotype was observed in rnpA+ bacteria which harbored a plasmid carrying a P7 c4 mutant gene with a single C-->G base substitution in the structural homologue to the CCA 3' end of tRNAs. Implications of C4 RNA processing for the lysis/lysogeny decision process of bacteriophages P1 and P7 are discussed.

Bacteriophage P1↗

PubMed1995

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.

Base Sequence↗

PubMed1995

Kinetics and thermodynamics of the RNase P RNA cleavage reaction: analysis of tRNA 3'-end variants.

We have studied the interaction of 3'-end variants of a (pre-)tRNAGly with ribonuclease P (RNase P) RNAs from Escherichia coli and Thermus thermophilus. To dissect the thermodynamics of tRNA binding from the overall catalytic reaction, specific binding of mature tRNAGly variants to RNase P RNAs was studied by gel retardation. A newly developed assay, based on the reduction of Pb(2+)-hydrolysis at the CCA end due to complex formation of tRNA and RNase P RNA, was utilized to confirm the dissociation constants. The binding data were supplemented by single and multiple turnover kinetic analyses of the corresponding pre-tRNAGly variants. For E. coli RNase P RNA the following results were obtained. Extensions of CCA by pCp or three nucleotides (AUA) stabilized gel-resolved tRNAGly binding by 1 to 1.5 kcal/mol. Changing the first C in CCA to A, G or U resulted in a more than 100-fold reduction in binding affinity, which corresponds to a loss of 3.5 to 4.5 kcal/mol of binding energy. However, single turnover rate constants were only slightly affected, indicating that a disruption or loss of the tRNA 3'-end-mediated interaction with RNase P RNA does not preferentially destabilize the transition state. Our data suggest another kinetic step following initial substrate binding to E. coli RNase P RNA (possibly a conformational rearrangement). For T. thermophilus RNase P RNA, product release of wild-type tRNAGly CCAAUA was not rate-limiting in the multiple turnover reaction. However, the effects of CCA mutations were similar to those attained with E. coli RNase P RNA. This supports the notion that a high-affinity binding site for the tRNA 3'-end is a ubiquitous feature of eubacterial P RNAs. Finally, the results obtained here provide further evidence that the gel retardation assay is suitable for binding interference studies to identify the structural elements of RNase P RNAs and tRNAs that are crucial for the formation of a specific RNase P RNA-tRNA complex.

Base Sequence↗

PubMed1995

Towards a new concept of gene inactivation: specific RNA cleavage by endogenous ribonuclease P.

In the first part of this chapter, general concepts for gene inactivation, antisense techniques and catalytic RNAs (ribozymes) are presented. The requirements for modified oligonucleotides are discussed with their effects on the stability of base-paired hybrids and on resistance against nuclease attack. This also includes the problems in the choice of an optimal target sequence within the inactivated RNA and the options of cellular delivery systems. The second part describes the recently introduced antisense concept based on the ubiquitous cellular enzyme ribonuclease P. This system is unique, since the substrate recognition requires the proper tertiary structure of the cleaved RNA. General properties and possible advantages of this approach are discussed.

Animals↗

PubMed1995

Point mutations in the leader boxA of a plasmid-encoded Escherichia coli rrnB operon cause defective antitermination in vivo.

We have introduced point mutations into the leader boxA of a plasmid-encoded Escherichia coli rrnB operon to study the in vivo role of this regulatory element in the natural context of rRNA synthesis. The same mutations were previously shown to cause severe antitermination defects in vitro and in the context of a reporter gene assay. The plasmid-encoded rrnB mutant constructs studied here also contained point mutations in the 16S and 23S rRNA genes, which were used to distinguish rRNAs derived from plasmid and chromosomal rrn operons by primer extension analysis. Point mutations in boxA reduced the fraction of plasmid-derived rRNA in the cell from 75% to about 50%. The reduction was similar for both 30S and 50S subunits as well as 70S ribosomes, suggesting that no transcriptional polarity occurred between the expression of the 16S and 23S rRNA genes in plasmid rrnB operons carrying a mutant boxA. The boxA mutations do not affect the amount of transcription initiation, suggesting that a suboptimal leader boxA causes premature transcription termination at an early stage of transcription. Our results are consistent with a role for antitermination in the completion of full-length rrn transcripts but give no indications of posttranscriptional boxA functions.

Base Sequence↗

PubMed1994

Contribution of structural elements to Thermus thermophilus ribonuclease P RNA function.

We have performed a deletion and mutational analysis of the catalytic ribonuclease (RNase) P RNA subunit from the extreme thermophilic eubacterium Thermus thermophilus HB8. Catalytic activity was reduced 600-fold when the terminal helix, connecting the 5' and 3' ends of the molecule, was destroyed by deleting 15 nucleotides from the 3' end. In comparison, the removal of a large portion (94 nucleotides, about one quarter of the RNA) of the upper loop region impaired function only to a relatively moderate extent (400-fold reduction in activity). The terminal helix appears to be crucial for the proper folding of RNase P RNA, possibly by orientating the adjacent universally conserved pseudoknot structure. The region containing the lower half of the pseudoknot structure was shown to be a key element for enzyme function, as was the region of nucleotides 328-335. Deleting a conserved hairpin (nucleotides 304-327) adjacent to this region and replacing the hairpin by a tetranucleotide sequence or a single cytidine reduced catalytic activity only 6-fold, whereas a simultaneous mutation of the five highly conserved nucleotides in the region of nucleotides 328-335 reduced catalytic activity by > 10(5)-fold. The two strictly conserved adenines 244 and 245 (nucleotides 248/249 in Escherichia coli RNase P RNA) were not as essential for enzyme function as suggested by previous data. However, additional disruption of two helical segments (nucleotides 235-242) adjacent to nucleotides 244 and 245 reduced activity by > 10(4)-fold, supporting the notion that nucleotides in this region are also part of the active core structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1994

Lead-ion-induced cleavage of RNase P RNA.

Pb(2+)-induced hydrolysis of RNase P RNAs from Escherichia coli and the thermophilic eubacterium Thermus thermophilus HB8 revealed one prominent site-specific cleavage in the two RNAs and several minor cleavage sites in structurally corresponding regions of both RNAs. Data presented here and in a previous study [Kazakov, S. & Altman, S. (1991) Proc. Natl Acad. Sci. USA 88, 9193-9197] provide evidence for several ubiquitous metal-ion-binding sites in eubacterial RNase P RNA subunits. With the T. thermophilus RNase P RNA, susceptibility to Pb(2+)-induced strand scission at the most prominent site was hypersensitive at the temperature of highest enzyme activity (55 degrees C). Pb2+ hydrolysis at this site was strongly reduced at a temperature of 37 degrees C, where processing is also inefficient. For E. coli RNase P RNA, specific changes in the lead hydrolysis pattern were observed due to the presence of excess tRNA. Thus, Pb(2+)-induced hydrolysis seems suitable to sense different conformations of RNase P RNAs. The T. thermophilus RNase P RNA, in particular, displayed significant processing activity after severe fragmentation by Pb2+, and therefore appears to be suited for reconstituting an active enzyme from RNA subfragments.

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↗

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

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

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↗

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

Analysis of the spc ribosomal protein operon of Thermus aquaticus.

The gene region of Thermus aquaticus corresponding to the distal portion of the S10 operon and to the 5'-portion of the Escherichia coli spc operon was cloned, using the E. coli gene for the ribosomal protein L5 as hybridization probe. The gene arrangement was found to be identical to E. coli, i.e. S17, L14, L24, L5, S14, S8 and L6. Stop and start regions of contiguous cistrons overlap, except for the S14-S8 intergenic region, whose size (67 bases) even exceeds the corresponding spacer regions in E. coli and Bacillus subtilis. A G + C content of 94% in third positions of codons was found in the ribosomal protein genes of T. aquaticus analyzed here. The stop codon of gene S17 (the last gene of the S10 operon in E. coli) and the start codon of gene L14 (the first gene of the spc operon in E. coli) overlap in T. aquaticus, thus leaving no space to accommodate an intergenic promoter preceding spc-operon-encoded genes in T. aquaticus. A possible promoter, localized within the S17 coding region, yielded only weak resistance (20 micrograms/ml) to chloramphenicol in E. coli and therefore could be largely excluded as the main promoter for spc-operon-encoded genes. We failed to detect a structure resembling the protein S8 translational repressor site, located at the beginning of the L5 gene in E. coli, in the corresponding region or any other region in the cloned T. aquaticus spc DNA.

Base Sequence↗