Changes in the promoter range of RNA polymerase resulting from bacteriophage T4-induced modification of core enzyme.

Abstract

Primary transcripts made in vitro on bacteriophage T4 DNA by RNA polymerase isolated from normal or T4-infected Escherichia coli were compared by gel electrophoresis. Bacteriophage-modified RNA polymerase fails to initiate transcription at certain promoters recognized by unmodified enzyme. In the T4 tRNA gene region, only one ofthe two promoters is active with the modified RNA polymerase. Reconstitution of separated RNA polymerase components demonstrates that this change in promoter site selection results from the modification of core enzyme and not a factor.During the development of bacteriophage T4 in its host Escherichia coli, complex changes occur in the transcription process. These changes include the shutoff of host transcription and sequential expression of three classes of bacteriophage genes, served by "early," "middle," and "late" promoters. The host RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) is apparently used for all transcription throughout bacteriophage infection, and it is generally believed that at least some ofthe changes in the transcription pattern are achieved through phage-induced changes in RNA polymerase specificity (for review, see ref. 1).Several modifications of RNA polymerase have been observed after T4 infection. They include chemical modification of the existing RNA polymerase subunits (2-13) and association with the enzyme of four small T4-coded polypeptides (14)(15)(16)(17)(18)(19)(20). Two of these new polypeptides (Mr 12,000 and 22,000) are the products ofT4 genes 33 and 55, which are required for the transcription ofthe late genes (15,16,18). No relationship has been established between other T4-induced RNA polymerase modifications and switches in the transcription pattern.Clearly, progress in understanding the molecular mechanisms of T4 transcription control depends on the development of in vitro systems that link particular RNA polymerase modifications with changes in transcription selectivity. Purified E. coli RNA polymerase in vitro recognizes only early promoters (21-23). In vitro transcription from middle (24) and late (25,26) promoters has been demonstrated only in crude lysates of T4-infected cells. The study of functional changes in T4-modified RNA polymerase by using purified transcription systems has thus far produced limited information. The modified enzyme competes poorly with the host RNA polymerase for template DNA (27), has a higher transition temperature for rapidly initiating transcription complexes (28), and is inhibited by 0.2 M KC1 (16). In contrast, the activity of the host RNA polymerase is markedly stimulated by KC1. The salt sensitivity of the modified enzyme is caused by a T4-coded polypeptide (Mr 10,000) that is associated with oa factor. RNA polymerase containing this protein fails to initiate transcription on T4 DNA at 0.2 M salt; this effect, however, can be overcome if 1% Triton X-405 is present at the moment of initiation (19,20). It has been shown that modified RNA polymerase transcribes certain bac...

The bacteriophage T4 anti‐sigma factor AsiA is not necessary for the inhibition of early promoters in vivo

“…It has long been known that purified T4‐modified RNA polymerase is much less efficient than the unmodified enzyme in transcribing bacteriophage T4 DNA in vitro , suggesting that one or more of these modifications is the cause of the early gene shut‐off. A number of studies aimed at understanding the molecular basis of this change in polymerase activity have involved core‐associated (ADP ribosylation and the RpbA protein) as well as sigma‐associated (AsiA) modifications (Seifert et al ., 1969; Khesin et al ., 1972; Stevens and Rhoton, 1975; Khesin et al ., 1976; Goldfarb, 1981; Goldfarb and Palm, 1981; Malik and Goldfarb, 1984; Drivdahl and Kutter, 1990). Our analysis with the double mutants shows that eliminating AsiA together with RpbA, Mod, MotB or MotA is without consequence for early gene shut‐off.…”

Distal regulation of alternative splicing by splicing enhancer in equine β-casein intron 1

“…Genomic DNA was isolated from the blood of 21 Lipizzan mares (of 30 mares for which splicing patterns were examined) according to Goldfarb (1981). Polymorphisms were detected by sequencing the fragments amplified in the promoter, intron 1, introns flanking the weak exons, and 3¢ untranslated region (UTR) of equine b-casein gene.…”

Abortive Infection by Bacteriophage Me1 of Escherichia coli K12 Strains Bearing the Plasmid ColV, I-K94

“…Native E. coli RNA polymerase cannot transcribe late phage genes until modified by several early phage proteins (Goldfarb, 1981). Furthermore, late mRNA is only produced from DNA that has recently replicated (Riva et al, 1970a).…”