Identification and characterization of the Escherichia coli rbn gene encoding the tRNA processing enzyme RNase BN.

The gene encoding RNase BN was localized to 88 min on the Escherichia coli chromosome by a novel suppressor assay and conjugational and transductional analysis. Assay of subclones derived from lambda phage 543 of the Kohara library, which encompasses this region of the chromosome, for elevated RNase BN activity identified o290, a previously reported open reading frame, as the gene encoding RNase BN. Interruption of this gene with a Kan(r) cassette and introduction into the chromosome eliminated cellular RNase BN activity but had no effect on cell growth. On the basis of these data, we suggest that o290 be renamed rbn. Potential homologs of rbn in other organisms also were identified.     

Cloning, characterization, and effects of overexpression of the Escherichia coli rnd gene encoding RNase D.

RNase D is a 3'-exoribonuclease whose in vitro specificity has suggested that it is involved in the processing of tRNA precursors. Its in vivo role has remained unclear, however, because mutant cells devoid of the enzyme display no defect in growth or tRNA processing. To learn more about the structure and function of RNase D, we cloned the Escherichia coli rnd gene, which is thought to code for this enzyme. The rnd gene was isolated from a cosmid library based on elevated RNase D activity and was subcloned as a 1.4-kilobase-pair fragment in pUC18. Maxicell analysis of the cloned fragment revealed that a single protein of approximately 40 kilodaltons, which is the size of RNase D, was synthesized. The rnd gene is present as a single copy on the E. coli chromosome and is totally absent in a deletion mutant. Cells that harbored the cloned rnd gene displayed RNase D activity that was elevated as much as 20-fold over that of the wild type. As growth of the culture progressed, however, RNase D specific activity declined dramatically, together with a similar decrease in plasmid copy number. In contrast, no decrease in copy number was observed with an inactive rnd gene. Placement of the rnd gene downstream from the lac promoter led to inducible RNase D overexpression and concomitantly slowed cell growth. These findings support the idea that rnd is the structural gene for RNase D and indicate that elevated RNase D activity is deleterious to E. coli.     

Escherichia coli RNase D. Purification and structural characterization of a putative processing nuclease

RNase D, a putative tRNa processing nuclease, has been purified about 1,000-fold from extracts of Escherichia coli to apparent homogeneity, as judged by acrylamide gel electrophoresis under nondenaturing and denaturing conditions and by gel electrofocusing. The purified enzyme is a single chain protein with a molecular weight of 40,000 and an isoelectric point of about 6.2. Spectral analysis indicated that RNase D is devoid of nucleic acid. Amino acid analysis suggested a low content of cysteine, and this was confirmed by the relative insensitivity of the enzyme to sulfhydryl group reagents. RNase D is sensitive to inactivation by elevated temperatures but can be protected by a variety of RNAs, including those which are not substrates for hydrolysis. The relation of RNase D to other known E. coli ribonucleases and to other previously identified processing activities, is discussed.     

Escherichia coli RNase D: sequencing of the rnd structural gene and purification of the overexpressed protein.

We have determined the nucleotide sequence of a 1.4-kb-pair fragment of the E. coli chromosome that carries the complete rnd gene encoding RNase D, a putative tRNA processing enzyme. The coding region of rnd extends for a total of 1128 nucleotides beginning at an initiator UUG codon and terminating at a UAA codon, and encodes a 375-amino acid polypeptide of 42,679 daltons, consistent with the known size of RNase D. A rapid purification procedure was developed for isolation of RNase D from strains overexpressing the enzyme. The N-terminal sequence and the amino acid composition of the homogenous protein were in excellent agreement with those derived from the sequence of the rnd gene.     

RNase E, an RNA processing enzyme from Escherichia coli.

An activity, RNase E, was purified about 100-fold from Escherichia coli cells, it can process p5 rRNA from a 9 S RNA molecule which accumulates in a mutant of E. coli defective in the maturation of 5 S rRNA. The enzyme requires Na+, K+, or NH4+, and Mg2+ or Mn2+. The molecular weight of the enzyme is about 70,000 and its pH optimum is 7.6 to 8.0. Its temperature optimum is around 30 degrees C, and it can be irreversibly inactivated at 50 degrees C. It has a very high degree of specificity but the reaction can be inhibited by nonspecific RNAs. We interpret its mode of action in producing p5 RNA as being accomplished in two steps, 9 S RNA is first processed to 7 S and 4 S, and subsequently 7 S is further processed to p5.     

RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells.

RNase PH is a Pi-dependent exoribonuclease that can act at the 3' terminus of tRNA precursors in vitro. To obtain information about the function of this enzyme in vivo, the Escherichia coli rph gene encoding RNase PH was interrupted with either a kanamycin resistance or a chloramphenicol resistance cassette and transferred to the chromosome of a variety of RNase-resistant strains. Inactivation of the chromosomal copy of rph eliminated RNase PH activity from extracts and also slowed the growth of many of the strains, particularly ones that already were deficient in RNase T or polynucleotide phosphorylase. Introduction of the rph mutation into a strain already lacking RNases I, II, D, BN, and T resulted in inviability. The rph mutation also had dramatic effects on tRNA metabolism. Using an in vivo suppressor assay we found that elimination of RNase PH greatly decreased the level of su3+ activity in cells deficient in certain of the other RNases. Moreover, in an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence. These data indicate that RNase PH can be an essential enzyme for the processing of tRNA precursors.     

A novel function of RNase P from Escherichia coli: processing of a suppressor tRNA precursor.

The leuX gene of Escherichia coli codes for a suppressor tRNA and forms a single gene operon containing its own promoter and Q-independent terminator. An analysis of the in vitro processing of leuX precursor revealed that the processing of the 5' end took place in a single-step reaction catalysed by RNase P while the 3' processing involved two successive reactions. The endonucleolytic cleavage activity of the 3' precursor sequence was found to copurify with RNase P. Heat inactivation of thermosensitive RNase P from two independent E. coli mutants abolished the cleavage activity of both the 5' and 3' ends. These results altogether suggest that RNase P carries the activity of 3' end cleavage as well as that of 5' processing. In the presence of Mg2+ alone, the leuX precursor was found to be self-cleaved at a site approximately 13 nt inside from the 5' end of mature tRNA. The self-cleaved precursor tRNA was no longer processed by the 3' endonuclease, suggesting that the 3' endonuclease recognizes a specific conformation of the precursor tRNA for action.     

RNase G-dependent degradation of the eno mRNA encoding a glycolysis enzyme enolase in Escherichia coli

Escherichia coli RNase G, encoded by the rng gene, is involved in the processing of 16S rRNA and degradation of the adhE mRNA encoding a fermentative alcohol dehydrogenase. In a search for the intracellular target RNAs of RNase G other than the 16S rRNA precursor and adhE mRNA, total cellular proteins from rng+ and rng::cat cells were compared by two-dimensional gel electrophoresis. The amount of enolase encoded by the eno gene reproducibly increased two- to three-fold in the rng::cat mutant strain compared with the rng+ parent strain. Rifampicin chase experiments showed that the half-life of the eno mRNA was some 3 times longer in the rng::cat mutant than in the wild type. These results indicate that the eno mRNA was a substrate of RNase G in vivo, in addition to 16S rRNA precursor and adhE mRNA.     

The rne gene is the structural gene for the processing endoribonuclease RNase E of Escherichia coli.

Summary Using T7 RNA polymerase and specific constructs derived from 5S rRNA and RNA I genes, we generated substrates for the RNA processing enzyme RNase E. Using these substrates we have shown that a 3.2 kb DNA fragment that complements the rne-3071 mutation can express RNase E activity. We also found that T7 RNA polymerase terminates within the 5S rRNA gene.     

Cloning, sequencing, and species relatedness of the Escherichia coli cca gene encoding the enzyme tRNA nucleotidyltransferase

The Escherichia coli cca gene which encodes the enzyme tRNA nucleotidyltransferase has been cloned by taking advantage of its proximity to the previously cloned dnaG locus. A series of recombinant bacteriophages, spanning the chromosomal region between the dnaG and cca genes at 66 min on the E. coli linkage map, were isolated from a lambda Charon 28 partial Sau3A E. coli DNA library using recombinant plasmids containing regions between dnaG and cca as probes. Two of the recombinant phage isolates, lambda c1 and lambda c4, contained the cca gene. A BamHI fragment from lambda c1 was subcloned into pBR328, and cells containing this recombinant plasmid, pRH9, expressed tRNA nucleotidyltransferase activity at about 10-fold higher level than the wild type control. The cca gene was further localized to a 1.4-kilobase stretch of DNA by Bal31 deletion analysis. The nucleotide sequence of the cca gene was determined by the dideoxy method, and revealed an open reading frame extending for a total of 412 codons from an initiator GTG codon that would encode a protein of about 47,000 daltons. Southern analysis using genomic blots demonstrated that the cca gene is present as a single copy on the E. coli chromosome and that there is no homology on the DNA level between the E. coli cca gene, and the corresponding gene in the Bacillus subtilis, Saccharomyces cerevisiae, Petunia hybrida, or Homo sapiens genomes. Homology was found only with DNA from the closely related species, Salmonella typhimurium. These studies have also allowed exact placement of the cca gene on the E. coli genetic map, and have shown that it is transcribed in a clockwise direction.     

Several regions of a tRNA precursor determine the Escherichia coli RNase P cleavage site.

The RNase P cleavage reaction was studied as a function of the number of base-pairs in the acceptor-stem and/or T-stem of a natural tRNA precursor, the tRNA(Tyr)Su3 precursor. Our data suggest that the location of the Escherichia coli RNase P cleavage site does not depend merely on the lengths of the acceptor-stem and T-stem as previously suggested. Surprisingly, we find that precursors with only four base-pairs in the acceptor-stem are cleaved by M1 RNA and by holoenzyme. Furthermore, we show that both disruption of base-pairing, and alteration of the nucleotide sequence (without disruption of base-pairing) proximal to the cleavage site result in aberrant cleavage. Thus, the identity of the nucleotides near the cleavage site is important for recognition of the cleavage site rather than base-pairing. The important nucleotides are those at positions -2, -1, +1, +72, +73 and +74. We propose that the nucleotide at position +1 functions as a guiding nucleotide. These results raise the possibility that Mg2+ binding near the cleavage site is dependent on the identity of the nucleotides at these positions. In addition, we show that disruption of base-pairing in the acceptor-stem affects both Michaelis-Menten constants, Km and kcat.     

Escherichia coli orfE (upstream of pyrE) encodes RNase PH.

RNase PH from extracts of Escherichia coli was purified to homogeneity and subjected to NH2-terminal sequencing. Comparison of this sequence with all open reading frames in the GenBank data base revealed at least 95% identity to an unidentified open reading frame (orfE) upstream of pyrE at 81.7 min on the E. coli chromosome. Clones of orfE overexpress RNase PH activity, verifying that orfE encodes this ribonuclease. We suggest that orfE be renamed rph.