Mapping and characterization of a mutation in Escherichia coli that reduces the level of ribonuclease III specific for double-stranded ribonucleic acid.

Localization of a mutation affecting ribonuclease III activity (an enzyme specific for double-stranded ribonucleic acid) in Escherichia coli was attempted. By a series of matings and transduction experiments, the mutation rnc-105 was mapped near the nadB gene. In strains carrying this mutation, another mutation (ranA2074) was also found. Based on available data, their order on the E. coli chromosome appears to be tyrA, ranA, nadB, rnc, purI. Strains carrying either the ranA2074 or the rnc-105 mutation fail to grow at 45 C in enriched medium, whereas strains carrying only the rnc-105 mutation are defective in ribonuclease III activity. Strains carrying either of these mutations grow more slowly than corresponding wild-type strains in all media tested at all temperatures; the rnc-105 mutation reduces the growth rate more than the ranA2074 mutation. T4 and T7 bacteriophages form plaques with a lower efficiency on strains carrying the rnc-105 mutation than on other strains. Thus we suggest that ribonuclease III is beneficial for normal growth of E. Coli and that at higher temperatures it becomes indispensable.     

Analysis of mRNA decay and rRNA processing in Escherichia coli multiple mutants carrying a deletion in RNase III.

RNase III is an endonuclease involved in processing both rRNA and certain mRNAs. To help determine whether RNase III (rnc) is required for general mRNA turnover in Escherichia coli, we have created a deletion-insertion mutation (delta rnc-38) in the structural gene. In addition, a series of multiple mutant strains containing deficiencies in RNase II (rnb-500), polynucleotide phosphorylase (pnp-7 or pnp-200), RNase E (rne-1 or rne-3071), and RNase III (delta rnc-38) were constructed. The delta rnc-38 single mutant was viable and led to the accumulation of 30S rRNA precursors, as has been previously observed with the rnc-105 allele (P. Gegenheimer, N. Watson, and D. Apirion, J. Biol. Chem. 252:3064-3073, 1977). In the multiple mutant strains, the presence of the delta rnc-38 allele resulted in the more rapid decay of pulse-labeled RNA but did not suppress conditional lethality, suggesting that the lethality associated with altered mRNA turnover may be due to the stabilization of specific mRNAs. In addition, these results indicate that RNase III is probably not required for general mRNA decay. Of particular interest was the observation that the delta rnc-38 rne-1 double mutant did not accumulate 30S rRNA precursors at 30 degrees C, while the delta rnc-38 rne-3071 double mutant did. Possible explanations of these results are discussed.     

The 3'' terminal oligonucleotide of E. coli 16S ribosomal RNA: the sequence in both wild-type and RNase iii- cells is complementary to the polypurine tracts common to mRNA initiator regions.

Application of Sanger techniques to the analysis of the 3' terminal oligonucleotide from E. coli 32-P-labelled 16 S rRNA yields the sequence AUCACCUCCUUAOH. This sequence is identical in RNA isolated from two wild-type strains (MRE600 and E. coli B, SY106) and from a mutant strain (AB301/105) defective in RNase III. Data presented here explains the previous derivation of an incorrect sequence (AUCCUCACUUCAOH) by others. The functional significance of complementarity between the 3' terminus of 16S rRNA and poly-purine tracts commonly found in mRNA initiator regions is discussed.     

Replication of RNA bacteriophages in the presence of rifamycin.

Replication of RNA bacteriophages in the presence of rifamycin was studied in different Escherichia coli strains that vary in RNase content but are not isogenic: AB259 RNase+, Q13 RNase I- PNPase-, AB105 RNase I- RNase III-. It was found that rifamycin did not affect characteristics of phage replication such as the general pattern of viral RNA synthesis and intracellular development of the phage. These characteristics are strain specific and independent of the cell growth rate, which defines only phage release. The inhibition of cell division by rifamycin interfered with the release of the phage and thus produced an apparent effect of rifamycin on phage replication.     

A conditional lethal mutant of Escherichia coli which affects the processing of ribosomal RNA.

A temperature-sensitive mutant strain was isolated from an RNase III-(rnc) strain of Escherichia coli. At the permissive temperature it behaves like the parental strain, but at the nonpermissive temperature it fails to produce normal levels of 23 S and 5 S rRNA, while instead the 25 S rRNA species becomes very prominent. (The 25 S molecule appears in rnc cells and contains 23 S rRNA sequences). When an rnc+ mutation was introduced to such a strain, or when the rnc mutation was replaced by an rnc+ allele, the strain remained temperature-sensitive. At the permissive temperature such strains synthesized rRNA like any other E. coli strain, but at the nonpermissive temperature they remained unable to synthesize normal levels of 5 S rRNA, and instead a larger molecule was accumulated. The simplest interpretation of theses findings is that the mutant strain contains a temperature-sensitive processing endoribonuclease, RNase E, which normally introduces a cut in the growing rRNA chain somewhere between the 23 S and the 5 S rRNA cistrons. These findings help also to explain the nature and origin of the various rRNA species observed in RNase III- cells and add to our understanding of processing of ribosomal RNA in normal cells of Escherichia coli.     

Revertants from RNase III negative strains of Escherichia coli

Summary E. coli strains carrying the rnc-105 allele do not show any level of RNase III in extracts, grow slower than rnc ⁺ strains at temperatures up to 45°C and fail to grow at 45°C. Revertants which can grow at 45°C were isolated. The vast majority of them still do not grow as fast as rnc ⁺ strains and did not regain RNase III activity. The mutation(s) which caused them are suppressor mutations (physiological suppressors) which do not map in the immediate vicinity of the rnc gene. A few of the revertants regain normal growth, and contain normal levels of RNase III. They do not harbor the rnc-105 allele and therefore are considered to be true revertants. By using purines other than adenine it was possible to isolate rnc ⁺ pur ^- revertants from an rnc ^- pur ^- strain with relative ease. They behaved exactly like the true rnc ⁺ revertants isolated from rnc ^- strains at 45°C.A merodiploid strain which contains the rnc ⁺ gene on an episome behaves exactly like an rnc ⁺ strain with respect to growth and RNA metabolism, eventhough its specific RNase III activity is about 60% of that of an rnc ⁺ strain; thus the level of RNase III is not limiting in the cell.The rnc ^- strains show a characteristic pattern of transitory molecules, related to rRNA, 30S, 25S, p23 and 18S, which are not observed in rnc ⁺ strains. This pattern is unchanged in rnc ^- strains and in the revertants which are still lacking RNase III, regardless of the temperature in which RNA synthesis was examined (30° to 45°C). On the other hand, in the rnc ⁺ strains as well as in the true revertants and the rnc ⁺/rnc ^- merodiploid, the normal pattern of p16 and p23 is observed at all temperatures. These findings suggest that all the effects observed in RNase III^- strains are due to pleiotropic effects of the rnc-105 allele, and that the enzyme RNase III is not essential for the viability of the E. coli cell.