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D Kennell

Publications and source records attributed to D Kennell.

At least 19 recordsLinked to original sources
PubMed1992

Broad-specificity endoribonucleases and mRNA degradation in Escherichia coli.

Crude extracts from Escherichia coli were screened for any broad-specificity endoribonuclease after the cell proteins were fractionated by size. In a mutant lacking the gene for RNase I (molecular mass, 27,156 Da), the only such activities were also in the size range of 23 to 28 kDa. Fractionation by chromatography on a strong cation-exchange resin revealed only two activities. One of them eluted at a salt concentration expected for RNase M and had the specificity of RNase M. It preferred pyrimidine-adenosine bonds, could not degrade purine homopolymers, and had a molecular mass of approximately 27 kDa (V. J. Cannistraro and D. Kennell, Eur. J. Biochem. 181:363-370, 1989). A second fraction, eluting at a higher salt concentration, was active against any phosphodiester bond but was about 100 times less active than are RNase I and RNase I* (a form of RNase I) in the wild-type cell. On the basis of sizing-gel chromatography, this enzyme had a molecular mass of approximately 24 kDa. We call it RNase R (for residual). RNase R is not an abnormal product of the mutant rna gene; a cell carrying many copies of that gene on a plasmid did not synthesize more RNase R. Our search for broad-specificity endoribonucleases was prompted by the expectation that the primary activities for mRNA degradation are expressed by a relatively small number of broad-specificity RNases. If correct, the results suggest that the endoribonucleases for this major metabolic activity reside in the 24- to 28-kDa size range. Endoribonucleases with much greater specificity must have as primary functions the processing of specific RNA molecules at a very limited number of sites as steps in their biosynthesis. In exceptional cases, these endoribonucleases inactivate a specific message that has such a site, and they can also effect total mRNA metabolism indirectly by a global disturbance of the cell physiology. It is suggested that a distinction be made between these processing and degradative activities.

Chromatography, Ion Exchange↗
PubMed1991

RNase I*, a form of RNase I, and mRNA degradation in Escherichia coli.

A previously unreported endoRNase present in the spheroplast fraction of Escherichia coli degraded homoribopolymers and small RNA oligonucleotides but not polymer RNA. Like the periplasmic endoRNase, RNase I, the enzyme cleaved the phosphodiester bond between any nucleotides; however, RNase I degraded polymer RNA as fast as homopolymers or oligomers. Both enzymes migrated as 27-kDa polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and could not be separated by various chromatographic procedures. In rna insertion mutants, both enzymes were completely missing; the spheroplast enzyme is called RNase I*, since it must be a form of RNase I. The two forms could be distinguished by physical treatments. RNase I could be activated by Zn2+, while RNase I* was inactive in the presence of Zn2+. RNase I was inactivated very slowly at 100 degrees C over a wide pH range, while RNase I* was inactivated slowly by heat at pH 4.0 but much more rapidly as the pH was increased to 8.0. In the presence of a thiol-binding agent, the inactivation at the higher pH values was much slower. These results suggest that RNase I*, but not RNase I, has free sulfhydryl groups. RNase I* activity in the cell against a common substrate was estimated to be several times that of RNase I. All four 2',3'-phosphomonoribonucleotides were identified in the soluble pools of growing cells. Such degradative products must arise from RNase I* activity. The activity would be suited for the terminal step in mRNA degradation, the elimination of the final oligonucleotide fragments, without jeopardizing the cell RNA. An enzyme with very similar specificity was found in Saccharomyces cerevisiae, suggesting that the activity may be widespread in nature.

Cytoplasm↗
PubMed1990

Cloning and sequencing the gene encoding Escherichia coli ribonuclease I: exact physical mapping using the genome library.

The amino acid (aa) sequence of the N terminus of Escherichia coli RNase I was determined. A mixed oligodeoxynucleotide coding for that sequence was used to probe the 476 lambda clones of Kohara et al. [Cell 50 (1987) 495-508]. DNA from these clones carry almost the entire E. coli chromosome in overlapping segments. Two overlapping clones hybridized to the probe sequence. From one of them DNA containing the rna gene was subcloned and sequenced. The inferred protein contains 245 aa residues and has an Mr of 27,156, which agrees with earlier estimates from sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. RNase I is close to twice the size of pancreatic RNase A, but both enzymes contain eight Cys and four His; those aa are important for structure and function of RNase A. Proximal to the rna gene is a sequence that would code for a 23-aa peptide which conforms to consensus rules for signal peptides, and thus should transport this periplasmic enzyme. Sites for eight restriction enzymes had been mapped on each lambda clone. By relating to the map for that specific region, it was possible to position the rna gene exactly at 659 kb from the thr locus (time zero on a time scale of 100 min). This physical mapping gave a more precise (exact) map position based on distance than was possible using genetic mapping based on a time scale derived from conjugation, and should be applicable for mapping many other E. coli genes.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of Escherichia coli RNase I. Comparisons with RNase M.

The endoribonuclease, RNase I, was purified from the periplasm of Escherichia coli. Based on PAGE, it has molecular mass of approximately 27 kDa with a migration rate indistinguishable from that of the recently reported RNase M from E. coli. The amino acid sequence of the two enzymes must be very similar based on two-dimensional mapping of their tryptic peptides and suggests either a post-transcriptional modification to yield different proteins from the same gene or evolution of two genes by gene duplication. However, while RNase I could degrade each of the four ribonucleotide homopolymers, only poly(U) or poly(C) were good substrates for RNase M with possibly some hydrolysis of poly(A). The reaction rate for poly(C) hydrolysis with RNase M was about ten times faster than for poly(U), while for RNase I the rates were about equal. Besides differences in specificity, RNase M was only located in the spheroplasts while RNase I found in the periplasm of growing cells. In terms of function, RNase I is known to cause degradation of rRNA during periods of stress or non-growth, whereas it has been proposed that RNase M is the endonuclease for mRNA degradation in growing cells.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of ribonuclease M and mRNA degradation in Escherichia coli.

A previously unreported endoribonuclease has been identified in Escherichia coli, which has a preference for hydrolysis of pyrimidine-adenosine (Pyd-Ado) bonds in RNA. It was purified about 7000-fold to give a single band after SDS/polyacrylamide gel electrophoresis; the eluted protein gave the same RNase specificity. The sizes of the native and denatured enzymes agreed suggesting that the enzyme exists as a monomer of approximately 26 kDa. It is called RNase M. The only other reported broadly specific endoribonuclease in E. coli is RNase I, a periplasmic enzyme. Based on differences in charge, heat stability and substrate specificity, it was clear that RNase M is not RNase I. The specificity of RNase M was remarkably similar to that of pancreatic RNase A even though the two enzymes differ in charge characteristics and size. Earlier studies had shown that mRNA from the lactose operon of E. coli is hydrolyzed in vivo primarily between Pyd-Ado bonds [Cannistraro et al. (1986) J. Mol. Biol. 192, 257-274] We propose that this major RNase activity accounts for these cleavages observed in vivo and that it is the endonuclease for mRNA degradation in E. coli.

Base Sequence↗
PubMed1988

Evidence for endonucleolytic cleavages in decay of lacZ and lacI mRNAs.

S1 nuclease mapping revealed lacZ mRNA molecules whose 5' and 3' ends were internal to the transcription start and consistent with cleavages at pyrimidine-adenosine bonds 20 to 50 nucleotides apart. With the net 5'-to-3' direction known, lacZ mRNA is probably degraded by sequential cleavages of naked mRNA at vulnerable sites exposed by transit of the last translating ribosome.

Bacterial Proteins↗
PubMed1986

Specific endonucleolytic cleavage sites for decay of Escherichia coli mRNA.

The polycistronic lac mRNA of Escherichia coli contains three messages. The rate of degradation of the second (lacY) message was observed to be equal to that of the third (lacA), and each decayed twice as fast as did the first (lacZ). Specific 5'- and 3'-ended lacY mRNA molecules could be recovered from cells; most likely, they are generated from endonucleolytic cleavages that are a part of the degradative process. They were observed by S1 nuclease mapping, and the exact 5'- and 3'-end oligonucleotides of many of them were identified by direct sequencing. Almost all of the molecules started with a 5' adenosine that would be preceded by a pyrimidine. The specificity was further restricted by neighboring nucleotides, and analysis of the data suggested that 5'-U-U decreases-A-U- is especially vulnerable. Also, computer analyses predicted the most stable secondary structures of selected segments of the mRNA and suggested that cleavages may only occur in regions of single strandedness. A model of mRNA degradation is proposed based on these observations and earlier ones. There is no unique target on a message for the initial inactivating attack: any region free of ribosomes is vulnerable, but for statistical reasons the initial attack of most molecules is near the ribosome-loading site. With no further ribosome loading, the newly unprotected 5' ends are "chopped off" at one of the next preferred target sites almost as fast as the last ribosomes moves down the mRNA.

Base Sequence↗
PubMed1985

The 5' ends of Escherichia coli lac mRNA.

We identified the predominant 5' ends of an mRNA in Escherichia coli to the exact nucleotides. There are four such ends of lac mRNA in fully induced cells. About 70% of the molecules have the reported major in vitro end, A-A-U-U-G (at +1), which is located 38 nucleotides before the A-U-G translation start. Another 15% start with A-U-U-G at +2, and about 8% start with A-U-U-A-G at -52. A fourth class of molecules begin with either A-G, C-A-G, A-C-A-G, or a weak A-C-A-C-A-G (at +24), observed only once. The origins of this latter set (less than or equal to 10% of the total) are not known, but they could represent "ragged" ends of the mRNA when it is degraded to the beginning of the ribosome-protected region of the message. The A-U-U-A-G molecules are probably initiated from an upstream promoter whose position would coincide with the cAMP-CRP DNA binding site for the major promoter.

Amino Acid Sequence↗
PubMed1985

Evidence that the 5' end of lac mRNA starts to decay as soon as it is synthesized.

By monitoring the decay of the first 16% of the beta-galactosidase message, we showed that the 5' end started to decay before the 3' end was completed and at a rate equivalent to that of the whole molecule. Thus, decay was neither from 3' to 5' nor from random internal fragmentation but rather proceeded in a net 5' to 3' direction.

Escherichia coli↗
PubMed1978

Translation and mRNA decay.

Degradation of messenger RNA from the lactose operon (lac mRNA) was measured during the inhibition of protein synthesis by chloramphenicol (CM) or of translation-initiation by kasugamycin (KAS). With increasing CM concentration mRNA decay becomes slower, but there is no direct proportionality between rates of chemical decay and polypeptide synthesis. During exponential growth lac mRNA is cleaved endonucleolytically (Blundell and Kennell, 1974). At a CM concentration which completely inhibits all polypeptide synthesis this cleavage is blocked. In contrast, if only the initiation of translation is blocked by addition of KAS, the cleavage rate as well as the rate of chemical decay are increased significantly without delay. These faster rates do not result from immediate degradation of the lengthening stretch of ribosome-free proximal message, since the full-length size is present and the same discrete message sizes are generated during inhibition. These results suggest that neither ribosomes nor translation play an active role in the degradative process. Rather, targets can be protected by the proximity of a ribosome, and without nearly ribosomes the probability of cleavage becomes very high. During normal growth there is a certain probability that any message is in such a vulnerable state, and the fraction of vulnerable molecules determines the inactivation rate of that species.

Aminoglycosides↗
PubMed1978

Altered mRNA metabolism in ribonuclease III-deficient strains of Escherichia coli.

The metabolism of mRNA from the lactose (lac) operon of Escherichia coli has been studied in ribonuclease (RNase) III-deficient strains (rnc-105). The induction lag for beta-galactosidase from the first gene was twice as long, and enzyme synthesis was reduced 10-fold in one such mutant compared with its isogenic rnc+ sister; in the original mutant strain AB301-105, synthesis of beta-galactosidase was not even detectable, although transduction analysis revealed the presence of a normal lac operon. This defect does not reflect a loss of all lac operon activity galactoside acetyltransferase from the last gene was synthesized even in strain AB301-105 but at a rate several times lower than normal. Hybridization analyses suggested that both the frequency of transcription initiation and the time to transcribe the entire operon are normal in rnc-105 strains. The long induction lag was caused by a longer translation time. This defect led to translational polarity with reduced amounts of distal mRNA to give a population of smaller-sized lac mRNA molecules. All these pleiotropic effects seem to result from RNase III deficiency, since it was possible to select revertants to rnc+ that grew and expressed the lac operon at normal rates. However, the rnc-105 isogenic strains (but not AB301-105) also changed very easily to give a more normal rate of beta-galactosidase synthesis without regaining RNase III activity or a faster growth rate. The basis for this reversion is not known; it may represent a "phenotypic suppression" rather than result from a stable genetic change. Such suppressor effects could account for earlier reports of a noninvolvement of RNase III in mRNA metabolism in deliberately selected lac+ rnc-105 strains. The ribosomes from rnc-105 strains were as competent as ribosomes from rnc+ strains to form translation initiation complexes in vitro. However, per mass, beta-galactosidase mRNA from AB301-105 was at least three times less competent to form initiation complexes than was A19 beta-galactosidase mRNA. RNase III may be important in the normal cell to prepare lac mRNA for translation initiation. A defect at this step could account for all the observed changes in lac expression. A potential target within a secondary structure at the start of the lac mRNA is considered. Expression of many operons may be affected by RNase III activity; gal and trp operon expressions were also abnormal in RNase III- strains.

Enzyme Induction↗