Bacteriophage f1 infection of Escherichia coli: identification and possible processing of f1-specific mRNAs in vivo.

Abstract

ABSTRACT[3HJUracil-pulse-labeled RNA from Eschenichia coli infected with fl bacteriophage was fractionated on polyacrylamide gels containing urea. Eight phage-specific RNA species were present with approximate lengths ranging from 2100 to 400 nucleotides. The amount of the seven largest species was increased when the infected bacteria were incubated at 420C. When the RNA was isolated and used as message in an in vitro protein-synthesizing system, most of the RNA species appeared to direct the synthesis of the phage gene VIII protein.The six largest species also directed the synthesis of the phage gene V protein. Some of the labeled smaller RNA species increased in amount after addition of rifampicin, suggesting that they may have resulted from cleavage of larger RNA species. These particular smaller RNA species also were present in infected bacteria containing a mutant RNase III. The data are discussed in terms of the regulation of synthesis of the phagespecific proteins.The closely related filamentous bacteriophage f1, fd, and M13 contain a single-stranded DNA that codes for approximately eight genes (1, 2). Escherichia coli infected with these phage continue to grow and divide, while newly synthesized phage are extruded through the membrane. The identification of most phage products has been hampered by this continuation of host growth. However, products of the phage genes V and VIII (coat protein) are synthesized in large quantities and can be easily identified (3, 4). The two proteins are also the major products of coupled transcription-translation directed in vitro by replicative form DNA (5, 6). These findings suggest that there is some regulatory mechanism operating both in vivo and in vitro that allows this differential expression of phage products.Such a regulatory process may be operating partially at the level of transcription. Studies in vitro have shown that a small number of discrete transcripts are synthesized; initiation is from various promoters, while termination appears to occur at a unique site (7-11). Due to their location in the genome (12), the gene V and VIII regions are immediately proximal to the termination site; thus, most mRNAs in vitro would contain the coding information for these genes.In order to examine the basis for the high level of expression of the gene V and VIII proteins in vivo, it is necessary to characterize phage-specific mRNA found in infected bacteria. We report here the identification of a number of phage-specific mRNAs and show that several of them can direct the synthesis of the gene V and VIII proteins. Some of the species appear to correspond to those found in vitro, while others may have arisen as a result of processing from larger RNA molecules. These results are discussed in terms of the regulation of synthesis of the phage-specific proteins.MATERIALS AND METHODS E. coli K38, a nonsuppressing strain, and K37, a serine-inserting suppressing strain, were used (13). BL214, an RNase III-strain (14), was obtained from W. Studier. Bacteriophage f1, fi amber mutants...

Ff coliphages: structural and functional relationships.

“…Consequently, genes located in front of the central terminator are transcribed more frequently than those at greater distances. This hypothesis is derived from and consistent with the following experimental findings: the viral genome contains sections of frequent as well as rare transcriptional activity (297), the former being congruent with the genes for proteins needed in high amounts (genes 5 and 8); in vitro transcription is initiated at several active (G) promoters, but terminated at only one site, the rho-independent T0.25 (which is also active in vivo) (36,37,297); and expression of genes 5 and 8 seems to be regulated mainly via the amount of corresponding mRNA synthesized.…”

“…An activity in the infected cell has only been demonstrated for promoters G0.92, G0.06 G0.12 G0.19 X0.25, and Ao.64, preceding genes 2, X, 5, 8, 3, and 4, respectively, and for the terminators T0.25 and hairpin A (261, 296, 297; van Wezenbeek, Ph.D. thesis). Cashman and Webster found eight phage specific mRNAs in fl-infected cells (36,37). Smits et al (297) detected at least 11 species in vivo.…”

Differential decay of a polycistronic Escherichia coli transcript is initiated by RNaseE‐dependent endonucleolytic processing

“…In phage T4 a precursor transcript of gene 32 was shown to be processed to a stable mRNA in an RNaseE-dependent manner (Mudd et ai, 1988). The expression of several genes in the filamentous bacteriophages f1 and Ml 3 includes post-transcriptional processing events that generate a set of relatively stable, functional mRNA molecules (Cashman and Webster, 1979;Cashman etai, 1980;Smits etai, 1980;Blumer and Steege, 1984;. However, the enzymatic activities involved remain to be elucidated (Blumer and Steege, 1989).…”

Filamentous phage IKe mRNAs conserve form and function despite divergence in regulatory elements 1 1 Edited by M. Gottesman

“…In the case of genes V and VIII, deduced sequences have been veri®ed by the amino acid compositions of the proteins (Nakashima et al, 1981;Peeters et al, 1983). In the gene II-VIII region of the IKe genetic map, the positions of predicted promoters (*) and rho-independent terminator (t) are indicated in parentheses (Peeters et al, 1985 (Hill & Petersen, 1982) and predicted for IKe (Peeters et al, 1985) (Cashman & Webster, 1979;Cashman et al, 1980;LaFarina & Model, 1983;Blumer & Steege, 1984 (Peeters et al, 1985). (Peeters et al, 1985).…”

“…As additional substrates for RNase E have been discovered, however, agreement on the sequence and structural requirements has become more dif®-cult (McDowall et al, 1994). Recent ®ndings that RNase E is part of a degradosome including an ATP-dependent RNA helicase also complicate further the issue of its substrate speci®city (Miczak et al, 1996;Py et al, 1996) (Schaller et al, 1978);C, D, E, F, and G (Blumer & Steege, 1984;C* (Kokoska et al, 1990); and H (Blumer & Steege, 1984 and this work (Blumer et al, 1987;IveyHoyle & Steege, 1989;Endemann & Model, 1995 (Johnson & Walseth, 1979), [g-32 P]ATP, H]uridine were purchased from ICN; [a-32 P]CTP, ENHANCE and GeneScreen Plus were from (Cashman & Webster, 1979). Bacteriophage IKe and host strains bearing the N plasmid were obtained from R. Webster (Duke University): JE2571/N3 is leu thr/N3, and P90C/N3 is ara Á (lac pro) thi (Sun & Webster, 1986 (Hanahan, 1983) or electroporation (Dower et al, 1988), and their structures were veri®ed by dideoxy sequencing methods (Tabor & Richardson, 1987).…”