Initiation, processing and termination of ribosomal RNA from a hybrid 5 S ribosomal RNA gene in a plasmid.

Transformation of an RNA-processing mutant (rne, RNase E-) of Escherichia coli with a recombinant plasmid containing the promoter region of the ribosomal cluster rrnA and portions from the 3' region of the rrnD cluster results in the accumulation of the precursors to 5 S ribosomal RNAs at the permissive as well as that of two full-length transcripts and a processing intermediate at the nonpermissive temperature. The two full-length transcripts start from the two rrnA promoters, which are about 120 nucleotides apart. This plasmid, pJR3 delta, contains an intact 5 S rRNA gene and portions from the 16 S and 23 S rRNA genes. Analysis of the major plasmid-specific RNA species revealed that RNA molecules initiated in vivo from the first promoter (P1) start with pppA, while transcripts from the second promoter (P2) contain either pppG or pppC at their 5' ends. Termination occurs mainly at the first available termination site. Full-length transcripts initiated from both promoters are processed to precursors of 5 S rRNAs in vivo at the permissive temperature, but only about 20% of these transcripts are processed to mature 5 S rRNA. RNA1 and RNA2 (the transcripts initiated from P1 and P2, respectively) and RNA3 (an RNA-processing intermediate containing the entire 5 S region and the 3' end of the transcripts) can be cleaved in vitro by cell extracts of wild type strains resulting in precursor and mature 5 S rRNAs in a reaction that is RNase E dependent but not ribosome dependent. The 5' end of the processed 5 S rRNA can correspond to the 5' end of mature 5 S rRNA or it can contain one to three additional nucleotides.