A transcription map of a 5.12 kb region containing the cos ends of actinophage 4 3 1 was determined using RNA prepared from induced and uninduced cultures of the temperature-sensitive lysogen, Streptomyces coelicolor A3(2) (fl3lctsl). In induced cultures, RNA synthesis was detected only late in the lytic cycle. A late operon initiated downstream of the int gene on the righthand end of the genome traversed the cos ends and extended a t least 3.6 kb along the left-hand end. Shorter, possibly processed, mRNAs were also present. The map was superimposed on the DNA sequence of 2.8 kb of the region, part of which had been determined previously and part of which is presented here. The late-expressed transcripts contained a tRNA*-like gene and four ORFs (1,2,3 and 5) detected on the basis of codon bias. Analysis of the putative protein products showed that one of the ORFs could encode a lysis protein and at least one may be involved in DNA maturation. Transcription mapping of RNA from uninduced cultures demonstrated a 620 nt transcript, xRNAl, of ORF6. So far this is the only gene in #C31 found to be expressed right to left with respect to the standard map of &31; its function during lysogenic growth could not be deduced from database searches.
An operon expressed late in the lytic cycle of the Streptomyces temperate phage C31 was shown to be transcribed from an inducible promoter, lp (phage late promoter), which resembled the previously reported early promoters. mRNAs initiated at lp were processed at the 3 end (and possibly also the 5 end) of a tRNA Thr -like sequence, resulting in leaderless polycistronic mRNAs.Vectors developed from the temperate Streptomyces phage C31 are widely used to study the molecular genetics of these commercially important, antibiotic-producing bacteria (2,3,14,15). There is considerable scope for the further exploitation of the genetic components of C31, and it is one of our aims to design expression systems based on the regulation of the phage promoters. To date, two types of promoters from C31 have been characterized. (i) The immediate-early promoters are active in nonlysogens and in uninduced lysogens and must therefore be recognized by the host RNA polymerase (11,22,24,25,27). (ii) The phage-specific early promoters are active only after induction into the lytic cycle and presumably require the expression of at least one immediate-early phage gene, probably encoding a transcription factor such as sigma factor or an activator (10,12,27). Early promoters are characterized by a 21-bp consensus sequence located Ϫ9/Ϫ10 to Ϫ30/Ϫ31 bp from the transcription initiation point (10). Transcription of early genes was maximal at 10 min, and expression of a reporter gene fused to an early promoter peaked at 20 min after induction of a temperature-sensitive lysogen of Streptomyces coelicolor (10, 12). The purpose of this work was to characterize a late promoter.Late gene expression occurs after the initiation of phage DNA synthesis, i.e., at least 10 min into the lytic cycle (18). Late transcripts, detectable at 20 min and later during lytic growth, map to the 18-kbp region to the left of the repressor gene and overlapping the cos ends (26). A late operon is transcribed from the extreme right-hand end of the C31 genome, through cos and at least 3.6 kbp into the left-hand end (9, 26). Low-resolution S1 mapping using a 1.3-kbp probe that included the region shown in Fig. 1A demonstrated the presence of two major RNA 5Ј endpoints, 720 and 540 bp to the left of the KpnI restriction site adjacent to the right-hand cos end (9) (Fig. 1A). Analysis of the DNA sequence showed an 18 out of 21 bp match (at coordinates 173 to 194 according to reference 9) to the consensus early promoters, close to the position of the upstream 5Ј endpoint in the RNA (Fig. 1A). We show here that a DNA fragment containing this sequence, hereafter called lp (phage late promoter), confers late promoter activity.In order to demonstrate promoter activity, a DNA fragment containing the locations of the 5Ј endpoints of both mRNAs mapped by S1 nuclease and one containing only the position of the downstream endpoint (Fig. 1A) were amplified by PCR and inserted upstream of the promoterless xylE gene in pIJ4083 (5) to make pCHT304 and pCHO405, respectively. Primers 4.4 (5Ј T...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.