Recognition and cleavage signals for mRNA processing lie within local domains of the phage f1 RNA precursors

Abstract

In Escherichia coli infected with the filamentous phage f1, a number of the abundant phage mRNAs, species C-G, are products of post-transcriptional processing. The approach of cloning the phage sequences likely to include the processing signals in a plasmid under transcriptional control of the lambda PL promoter (Blumer, K. J., and Steege, D. A. (1984) Nucleic Acids Res. 12, 1847-1861) was extended to additional sites to show that processing at all five major sites is mediated by nuclease activity encoded by the bacterial genome. Primer extension methods were used to map more accurately in the f1 DNA sequence the 5' end points of the processed RNAs. The DNA segments that encode the mRNA processing signals were delimited from parallel series of 5'-3' and 3'-5' deletions made into the regions in which the RNA 5' ends map. For each deletion variant, in vivo f1 mRNA processing activity was assessed by primer extension and S1 nuclease mapping methods. The data indicate that the processing signals are comprised of relatively local regions near the point of RNA cleavage. Whereas cleavage occurs at the 5' border of the sequences that comprise the D, E, and F processing sites and thereby places most of the recognition information in the mature or product portion of the precursor, it occurs more centrally within the region comprising the C site. The C and D sites function independently as substrates for cleavage, with the necessary information contained in regions of 70 and 90 nucleotides, respectively. Cleavage at the E site appears to require a region of 130 nucleotides which completely contains the F site. From the effects on processing activity of deleting sequences in this region, the overlapping E and F processing sites appear to consist functionally of two subdomains. Each has a cleavage site at its immediate 5' end which can be substituted by foreign sequences, but both utilize a common recognition domain downstream from the point of strand scission.