To test whether any specific 5' precursor sequences are required for the processing of pre-16S rRNA, constructs were studied in which large parts of the 5' leader sequence were replaced by the coliphage lambda PL promoter and adjacent sequences. Unexpectedly, few full-length transcripts of the rRNA were detected after the pL promoter was induced, implying that either transcription was poor or most of the rRNA chains with lambda leader sequences were unstable. Nevertheless, sufficient transcription occurred to permit the detection of processing by Si nuclease analysis. RNA transcripts in which 2/3 of the normal rRNA leader was deleted (from the promoter up to the normal RNase III cleavage site) were processed to form the normal 5' terminus.Thus, most of the double-stranded stem that forms from sequences bracketing wild-type 16S pre-rRNA is apparently not required for proper processing; the expression of such modified transcripts, however, must be increased before the efficiency of processing of the 16S rRNA formed can be assessed.Synthesis of rRNA includes transcription of extensive leader, intercistronic, and trailer sequences that are discarded during the processing of pre-rRNA. The function of these sequences is not yet clear. Sequences within the leader region facilitate transcription (see below), and sequences 5'-proximal and 3'-distal to 16S rRNA pair to form a stable stem (reviewed in reference 15). The stem encloses the 16S rRNA in a large loop, and an analogous loop is formed by sequences that bracket the 23S rRNA. Because similar base-paired stems are conserved in bacteria as disparate as Escherichia coli, Bacillus subtilis, and an archaebacterium, these features must somehow be important (15).One suggestion has been that the double-stranded segments are important because endonucleolytic cuts in the stems, which occur early in processing, are required for the production of mature rRNA. That notion became less attractive, however, when accurate formation of 16S rRNA was observed in a mutant unable to cleave the doublestranded stem (13). As another possibility, precursor sequences might help to promote a conformation in 16S rRNA that is required for ribosome formation and forms more easily in the precursor RNA. Consistent with this speculation, pre-16S rRNA shows some features of secondary structure not found in the mature rRNA (16) and can bind certain ribosomal proteins more effectively than does mature rRNA (19).One way to examine the role of a precursor sequence is to eliminate or modify it by recombinant DNA manipulation and to determine the properties of the modified pre-rRNA in vivo. To investigate the possible requirement of 5' spacer sequences for the formation of 16S rRNA, we removed them from an rRNA operon in a high-copy-number plasmid. The promoter and, in different constructs, some or all of the sequence proximal to the 5' terminus of 16S rRNA were replaced by an inducible lambda promoter and lambda sequences. Such constructs can be used to distinguish among extreme alternatives. If processi...
Transcription of the bacteriophage X nutL region from the pL promoter on a multicopy plasmid in Escherichia coli causes a reduction in growth rate and in transcription of rRNA relative both to total transcription and to transcription of tRNAs that are not encoded in rRNA operons. These observations support the hypothesis, previously based on nut site DNA sequence homology, that the phage X and rRNA antitermination systems are related.
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