The ρ-dependent transcription terminator tR1 of bacteriophage λ stops RNA synthesis downstream of the major rightward promoter, PR, shortly after the cro gene. Terminated transcripts produced in a purified in vitro transcription system display a heterodisperse set of 3′ termini, occurring in clusters located at +290-300, 308–312, 340–345, 385–390, and 440–450 nucleotides from the transcription start site [Morgan, W. D., Bear, D. G., & von Hippel, P. H. (1983) J. Biol. Chem. 258, 9553–9564]. However, transcripts from the same promoter in vivo have been reported to end primarily at +310-312 [Court, D., Brady, C., Rosenberg, M., Wulff, D. L., Behr, M., Mahoney, M., & Izumi, S. (1980) J. Mol. Biol. 138, 231–254]. In order to understand the nature of this discrepancy, we have carried out a comparative analysis of λ PR transcription products producd in translationally active S30 cell extracts, in a purified in vitro system and in vivo. RNAs from the cell extracts coupled to translation show primarily three PR-derived transcripts beginning at one predominant 5′ end and terminating at +263, 308, and 318. Sites +263 and +308 appear to be RNA processing sites. S1 nuclease mapping studies of RNAs produced in vivo show one 5′ end and two 3′ termini ending at +263 and 311; the +263 site is the predominant 3′ end. When transcripts produced in a purified in vitro transcription system are incubated in the S30 cell extract under various conditions, the RNAs are degraded to two primary products with lengths of 263 and 308–311 nt. Processing of Cro transcripts to the 263- and 308-nt species is not observed in S30 extracts containing a defective RNase II enzyme and lacking polynucleotide Phosphorylase activity. These studies indicate that the 3′ termini of λ PR transcripts are formed by a combination of at least two processes: ρ-dependent transcription termination and RNA processing by 3′-exonucleolytic digestion.
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