ANALYSIS OF RIBONUCLEASE III FROM CAULOBACTER CRESCENTUS AND GENERATION OF A TN5-DERIVED PROMOTER-PROBE FOR STUDY OF GENE EXPRESSION IN THIS ORGANISM (TRANSPOSONS, RIBOSOMAL-RNA)
BELLOFATTO, VIVIAN MARIE
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An RNA processing enzyme has been isolated from Caulobacter crescentus which is specific for double-stranded RNA, has a requirement for monovalent and divalent cations and is eluted from a poly (I):poly (C) affinity column in pure form. This enzyme, like Escherichia coli RNase III, processes precursor ribosomal RNAs and polycistronic phage mRNAs and has a monomeric M(,r) of approximately 20,000. These two enzymes differ, however, in the recognition of specific cleavage sites and yield different digestion products when either coliphage T7 or C. crescentus phage OCdl early mRNA is used as substrate. It appears that an RNase III activity functions as a processing enzyme in C. crescentus since: (a) In an in vitro reaction, C. crescentus phage OCdl major early mRNA synthesized in vitro was processed by RNase III to yield RNA species which co-migrated with phage RNA synthesized in vivo, and (b) an in vitro transcript of a C. crescentus ribosomal RNA clone was processed by C. crescentus RNase III to yield an RNA product which co-migrated with 16S RNA.;A promoter-probe was constructed to study gene expression in Caulobacter. The promoter-probe, Tn5-VB32, was placed in an Inc P group plasmid which can be transferred to bacteria such as Caulobacter, Rhizobium and Agrobacterium. A fragment of DNA containing the NPT II gene from Tn5, lacking its promoter but retaining its translational initiation signals, was inserted into a Tn5 derivative that lacked the entire NPT II gene and a large portion of the IS5OL sequence while retaining its ability to transpose. The presence of the Tn5 derivative in the genome could be easily detected because this construct contained Tc('R) determinant of transposon Tn10. Transposition of the Tn5-VB32 promoter probe into the C. crescentus genome generated auxotrophic and mobility mutants. In these strains, Tn5-VB32 had inserted randomly into the chromosome. In a mutant strain containing the promoter-probe inserted into a gene involved in cysteine biosynthesis, the expression of the NPT II gene was shown to be under the regulation of exogenous cysteine. Several fla('-) mutants were isolated by Tn5-VB32 insertional inactivation and shown to confer varying levels of kanamycin resistance.