PubMed Health⌕ Search

Biomedical subjects

V R Kaberdin

Publications and source records attributed to V R Kaberdin.

14 recordsLinked to original sources
PubMed2001

Cleavage of poly(A) tails on the 3'-end of RNA by ribonuclease E of Escherichia coli.

RNase E initiates the decay of Escherichia coli RNAs by cutting them internally near their 5'-end and is a component of the RNA degradosome complex, which also contains the 3'-exonuclease PNPASE: Recently, RNase E has been shown to be able to remove poly(A) tails by what has been described as an exonucleolytic process that can be blocked by the presence of a phosphate group on the 3'-end of the RNA. We show here, however, that poly(A) tail removal by RNase E is in fact an endonucleolytic process that is regulated by the phosphorylation status at the 5'- but not the 3'-end of RNA. The rate of poly(A) tail removal by RNase E was found to be 30-fold greater when the 5'-terminus of RNA substrates was converted from a triphosphate to monophosphate group. This finding prompted us to re-analyse the contributions of the ribonucleolytic activities within the degradosome to 3' attack since previous studies had only used substrates that had a triphosphate group on their 5'-end. Our results indicate that RNase E associated with the degradosome may contribute to the removal of poly(A) tails from 5'-monophosphorylated RNAs, but this is only likely to be significant should their attack by PNPase be blocked.

3' Untranslated Regions↗
PubMed2000

Enhanced cleavage of RNA mediated by an interaction between substrates and the arginine-rich domain of E. coli ribonuclease E.

Endonucleolytic cutting by the essential Escherichia coli ribonuclease RNaseE has a central role in both the processing and decay of RNA. Previously, it has been shown that an oligoribonucleotide corresponding in sequence to the single-stranded region at the 5' end of RNAI, the antisense regulator of ColE1-type plasmid replication, is efficiently cut by RNaseE. Combined with the knowledge that alteration of the structure of stem-loops within complex RNaseE substrates can either increase or decrease the rate of cleavage, this result has led to the notion that stem-loops do not serve as essential recognition motifs for RNaseE, but can affect the rate of cleavage indirectly by, for example, determining the single-strandedness of the site or its accessibility. We report here, however, that not all oligoribonucleotides corresponding to RNaseE-cleaved segments of complex substrates are sufficient to direct efficient RNaseE cleavage. We provide evidence using 9 S RNA, a precursor of 5 S rRNA, that binding of structured regions by the arginine-rich RNA- binding domain (ARRBD) of RNaseE can be required for efficient cleavage. Binding by the ARRBD appears to counteract the inhibitory effects of sub-optimal cleavage site sequence and overall substrate conformation. Furthermore, combined with the results from recent analyses of E. coli mutants in which the ARRBD of RNase E is deleted, our findings suggest that substrate binding by RNaseE is essential for the normal rapid decay of E. coli mRNA. The simplest interpretation of our results is that the ARRBD recruits RNaseE to structured RNAs, thereby increasing the localised concentration of the N-terminal catalytic domain, which in turn leads to an increase in the rate of cleavage.

Amino Acid Motifs↗
PubMed2000

Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding.

The adaptation of mRNA stability to environmental changes is a means of cells to adjust the level of gene expression. The Escherichia coli ompA mRNA has served as one of the paradigms for regulated mRNA decay in prokaryotes. The stability of the transcript is known to be correlated inversely with the bacterial growth rate. Thus, the regulation of ompA mRNA stability meets the physiological needs to adjust the level of ompA expression to the rate of cell division. Recently, host factor I (Hfq/HF1) was shown to be involved in the regulation of ompA mRNA stability under slow growth conditions. Here, we present the first direct demonstration that 30S ribosomes bound to the ompA 5'-UTR protect the transcript from RNase E cleavage in vitro. However, the 30S protection was found to be abrogated in the presence of Hfq. Toeprinting and in vitro translation assays revealed that translation of ompA is repressed in the presence of Hfq. These in vitro studies are corroborated by in vivo expression studies demonstrating that the reduced synthesis rate of OmpA effected by Hfq results in functional inactivation of the ompA mRNA. The data are discussed in terms of a model wherein Hfq regulates the stability of ompA mRNA by competing with 30S ribosomes for binding to the ompA 5'-UTR.

5' Untranslated Regions↗
PubMed1998

The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly.

Escherichia coli RNase E, an essential single-stranded specific endoribonuclease, is required for both ribosomal RNA processing and the rapid degradation of mRNA. The availability of the complete sequences of a number of bacterial genomes prompted us to assess the evolutionarily conservation of bacterial RNase E. We show here that the sequence of the N-terminal endoribonucleolytic domain of RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria. Furthermore, we demonstrate that the Synechocystis sp. homologue binds RNase E substrates and cleaves them at the same position as the E. coli enzyme. Taken together these results suggest that RNase E-mediated mechanisms of RNA decay are not confined to E. coli and its close relatives. We also show that the C-terminal half of E. coli RNase E is both sufficient and necessary for its physical interaction with the 3'-5' exoribonuclease polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase, which are components of a "degradosome" complex. Interestingly, however, the sequence of the C-terminal half of E. coli RNase E is not highly conserved evolutionarily, suggesting diversity of RNase E interactions with other RNA decay components in different organisms. This notion is supported by our finding that the Synechocystis sp. RNase E homologue does not function as a platform for assembly of E. coli degradosome components.

Base Sequence↗
PubMed1998

RNA components of Escherichia coli degradosome: evidence for rRNA decay.

Recently, we found that a multicomponent ribonucleolytic degradosome complex formed around RNase E, a key mRNA-degrading and 9S RNA-processing enzyme, contains RNA in addition to its protein components. Herein we show that the RNA found in the degradosome consists primarily of rRNA fragments that have a range of distinctive sizes. We further show that rRNA degradation is carried out in the degradosome by RNase E cleavage of A+U-rich single-stranded regions of mature 16S and 23S rRNAs. The 5S rRNA, which is known to be generated by RNase E processing of the 9S precursor, was also identified in the degradosome, but tRNAs, which are not cleaved by RNase E in vitro, were absent. Our results, which provide evidence that decay of mature rRNAs occurs in growing Escherichia coli cells in the RNA degradosome, implicate RNase E in degradosome-mediated decay.

Chromatography, Affinity↗
PubMed1996

RNase E cleaves at multiple sites in bubble regions of RNA I stem loops yielding products that dissociate differentially from the enzyme.

Earlier work has shown that RNase E cleaves RNAI, the antisense repressor of replication of ColE1-type plasmids, producing pRNAI-5, whose further decay is mediated by the poly(A)-dependent activity of polynucleotide phosphorylase and other 3' to 5' exonucleases. Using a poly(A) polymerase-deficient strain to impede exonucleolytic decay, we show that RNAI is additionally cleaved by RNase E at multiple sites, generating a series of decay intermediates that are differentially retained by the RNA binding domain (RBD) of RNase E. Primer extension analysis of RNAI decay intermediates and RNase T1 mapping of the cleavage products of RNAI generated in vitro by affinity-purified RNase E showed that RNase E can cleave internucleotide bonds in the bubble regions of duplex RNA segments and in single-stranded regions. Chemical in situ probing of a complex formed between RNAI and the RBD indicates that binding to the RBD destabilizes RNAI secondary structure. Our results suggest a model in which a series of sequential RNase E-mediated cleavages occurring at multiple sites of RNAI, some of which may be made more accessible to RNase E by the destabilizing effects of its RBD, generate RNA fragments that are further degraded by poly(A)-dependent 3' to 5' exonucleases.

Base Sequence↗
PubMed1996

Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed.

Amino Acid Sequence↗
PubMed1996

Characterization of the structure and melting behavior of the loop I fragment of ColE1 RNA I.

We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5'-end, and a 31-mer which has 11 5'-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5' end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32 degrees C and 71 degrees C clearly discernible. In conjunction with NMR melting study the major transition at 71 degrees C is assigned to the overall melting of the stem region and the 32 degrees C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.

Base Composition↗
PubMed1995

Site-specific RNase E cleavage of oligonucleotides and inhibition by stem-loops.

The enzyme RNase E (ref. 1) cuts RNA at specific sites within single-stranded segments. The role of adjacent regions of secondary structure in such cleavages is controversial. Here we report that 10-13-nucleotide oligomers lacking any stem-loop but containing the RNase E-cleaved sequence of RNA I, the antisense repressor of replication of ColE1-type plasmids, are cut at the same phosphodiester bond as, and 20 times more efficiently than, RNA I. These findings indicate that, contrary to previous proposals, stem-loops do not serve as entry sites for RNase E, but instead limit cleavage at potentially susceptible sites. Cleavage was reduced further by mutations in a non-adjacent stem-loop, suggesting that distant conformational changes can also affect enzyme access. Modulation of RNase E cleavages by stem-loop regions and to a lesser extent by higher-order structure may explain why this enzyme, which does not have stringent sequence specificity, cleaves complex RNAs at a limited number of sites.

Base Sequence↗
PubMed1991

Simple approach for oligonucleotide-directed mutagenesis of any double-stranded circular DNA.

A new site-directed method for introducing mutations into any region of plasmid vector close to the unique restriction site is described. It is based on the use of 5'-phosphorylated mutagenic and nonphosphorylated auxiliary oligonucleotides and a specific combination of enzymatic procedures including 'nick-translation' as a key step. The method efficiency was demonstrated by constructing the deletion-insertion mutation which creates the consensus Pribnow box in a promoter-testing plasmid. The yield of the target mutation was up to 85-95%.

Base Sequence↗
PubMed1991

[A simple way of introducing a transcription initiation signal in vector DNA].

A new effective method of site-specific mutagenesis in the close vicinity of unique restriction sites of plasmids, based on the use of two oligonucleotide primers, mutagenic and auxiliary, has been suggested. A site-specific insertion of Pribnow box (TATAATG) before promoterless gal operon of the promoter-testing plasmid pHD-001-14-11 has been performed with the yield of mutants up to 95%. Data on the gal operon expression and S1-nuclease mapping of the transcription start point indicate the formation of an active promoter in the region of the insertion.

Base Sequence↗
PubMed1991

[An effective method for site-directed mutagenesis in plasmids and cloning single-stranded DNA fragments].

A three primer variant of the earlier devised oligonucleotide-directed mutagenesis in plasmids is described, useful also for the fast cloning of single-stranded DNA products of the asymmetric polymerase chain reaction (PCR). Using this method for plasmid pHD-001-14-11, a 59 b. p. deletion and a 7 b. p. insertion were simultaneously introduced at 81% frequency, and the PCR-copied phage fd transcription terminator (26 b. p.) was inserted with the yield of 67%.

Autoradiography↗