T4-modified RNA Polymerase Research Articles

Transcription from a bacteriophage T4 middle promoter using T4 motA protein and phage-modified RNA polymerase.

The bacteriophage T4 motA protein is required for transcription from T4 middle promoters. These promoters, which contain the Escherichia coli promoter consensus sequence at the -10 region (TATAAT) but a unique sequence centered at -30 ((a/t)(a/t)TGCTT(t/c)A) (Guild, N., Gayle, M., Sweeney, R., Hollingsworth, T., Modeer, T., and Gold, L. (1988) J. Mol. Biol. 199, 241-258), become active about 2 min after infection, a time when the host RNA polymerase has been modified by phage proteins. This paper shows that motA protein binds to a T4 middle promoter in vitro and that the addition of the motA protein allows in vitro transcription from this promoter by T4-modified RNA polymerase. The T4 motA gene was cloned into a multicopy plasmid that complemented T4 motA mutants in vivo. MotA protein, partially purified from cells containing a motA+ plasmid, specifically retarded the electrophoretic mobility of an oligomer containing the T4 middle promoter located 195 bases upstream of uvsX (PuvsX). RNA polymerase isolated from infected cells during T4 middle gene expression supported in vitro transcription from PuvsX only when fractions containing the motA protein were added. In contrast, unmodified host RNA polymerase catalyzed the synthesis of minor amounts of RNA from PuvsX, but this synthesis was not motA dependent. Thus, the in vitro transcription system described here provides the basis for a detailed study of the phage and host factors needed to regulate T4 middle gene expression.

Phage T4 DNA codes for two distinct 10-kDa proteins which strongly bind to RNA polymerase

Radioactive proteins have been synthesized in vitro from a coupled transcription-translation system primed with bacteriophage T4 DNA. The labeled proteins were chromatographed on RNA polymerase-Sepharose affinity columns in order to identify a protein which comigrates with the 10-kDa anti-σ subunit of T4-modified RNA polymerase. When we primed the in vitro system with specific restriction fragments of T4 DNA, we found that there seemed to be two widely separated genes which code for this protein. The products of these two genes were compared by two-dimensional electrophoresis; they were found to have different charges, even though they had the same molecular weight and strong affinity for RNA polymerase. One of these proteins exists in a form which has the same charge as the 10-kDa subunit isolated from purified T4-modified RNA polymerase. We report a preliminary mapping experiment within the 22.5-kb fragment harboring this gene.

Transcription at bacteriophage T4 variant late promoters. An application of a newly devised promoter-mapping method involving RNA chain retraction.

Bacteriophage T4 late promoters display a conserved sequence that extends over about 18 base pairs, in which the central 8-base pair sequence (TATAAATA in the nontranscribed strand) is absolutely conserved. Transcription by T4-modified RNA polymerase in vitro nevertheless initiates within a cluster of 6 overlapping variant T4 late promoters that deviate from the absolutely conserved sequence at one or two positions. One of these variant promoters is dominant, and its sequence implies that two of the absolutely conserved nucleotides (underlined above) are not essential. Three other variant promoters that contain only one of the deviations found in the dominant variant promoter are, at best, utilized weakly, suggesting that sequences outside the absolutely conserved segment are important for promoter function. A newly devised method, based on arrest of RNA chain elongation with ribonucleotide analogs and its reversal, has been used to precisely map initiation within the overlapping promoter cluster. Multiple cycles of RNA chain arrest, pyrophosphorolysis, and resynthesis can be executed. This process permits a precise stepwise control of the advance of a transcription complex through a gene.

Initiation of transcription by bacteriophage T4-modified RNA polymerase independently of host sigma factor

After infection of Escherichia coli with bacteriophage T4 a series of modifications of RNA polymerase takes place including the association of several small polypeptides. We isolated RNA polymerase from cells abortively infected with a series of T4 mutants which arrest phage development at different stages and found that different sets of associated proteins are present in RNA polymerase in each case. The patterns of associated polypeptides seem to correlate with DNA content in the infected cells, suggesting that some of them can be involved both in DNA replication and in the transcription apparatus. One of the modified forms of RNA polymerase contains stoichiometric amounts of a protein with M r = 25,000 (25K protein), which remains associated with the core enzyme after the removal of sigma factor by chromatography on phosphocellulose. The 25K protein was purified to homogeneity and its effect on transcription selectivity was analyzed in an in vitro system using fragments of T4 DNA as templates. The 25K protein exists in two functional forms which direct core RNA polymerase to utilize two different types of transcription start sites (class I and class II promoters). Both activities do not require host sigma factor. The two forms of 25K protein seem to compete with each other for the core enzyme. The isolated 25K protein can form stable dimers, suggesting that its two activities are associated with the dimeric and monomeric forms. Class I (but not class II) promoters can also be utilized in response to the host sigma factor. An hypothesis is proposed that T4-induced 25K protein serves as a new specificity factor with respect to class II sites and as a substitute for the host sigma with respect to the class I sites.

Changed promoter specificity and antitermination properties displayed in vitro by bacteriophage T4-modified RNA polymerase

A 5.5 × 10 3 base-pair fragment of bacteriophage T4 DNA carrying genes 1, 3, 57, ipI and a cluster of transfer RNA genes was used as template for RNA polymerase isolated from uninfected Escherichia coli and from T4-infected bacteria. RNA transcripts were fractionated by gel electrophoresis and mapped by using as transcription template the 5.5 × 10 3 base fragment cleaved with different restriction enzymes. The comparison of the transcripts synthesized by the two RNA polymerases revealed a dramatic difference in their initiation specificities and abilities to utilize a transcription termination site. The T4-modified polymerase utilizes three new promoters on the template DNA fragment that are not utilized by the host enzyme. The modified enzyme, however, fails to produce some of the transcripts synthesized by the host RNA polymerase. The ability of T4-modified RNA polymerase to terminate transcription at a terminator present in the template DNA fragment is greatly reduced as compared to the unmodified host enzyme. The factors responsible for the new initiation and termination properties are associated with RNA polymerase core component. Analysis of RNA polymerase from bacteria infected with T4 mutants demonstrates that the new promoter specificity and the antitermination effect are caused by different factors.

Initiation of transcription at phage T4 late promoters with purified RNA polymerase

We have previously identified T4 late promoters governing the in vivo expresson of T4 late genes 23 and 24 ( P 23 and P 24). T4 late transcription in vivo is known to involve the binding of at least five phagecoded proteins to the bacterial RNA polymerase and normally requires concurrent DNA replication for DNA template activation. We show here that in vitro transcription, primarily of plasmids carrying T4 genes 23 and 24, by RNA polymerase purified from Escherichia coli at late times after T4 infection allows specific initiation at P 23 and P 24 in the absence of DNA replication. These promoters are not utilized by E. coli RNA polymerase holoenzyme, by RNA polymerase core, or by T4-modified RNA polymerase purified from cells infected with a T4 gene 55 mutant (gene 55 codes for an RNA polymerase binding protein required for late transcription). The utilization of P 23 and P 24 in vitro is sharply inhibited by NaCI concentrations greater than 100 mM, and this inhibition is partly reversed by the addition of 10% DMSO. Relaxation of plasmid DNA containing P 23 (with topoisomerase I) reduces P 23 utilization at low salt (50 mM Na +) and nearly abolishes it at high salt (250 mM Na +). P 23 utilization is discernible in linear, glucosylated hydroxymethylcytosine-containing T4 virion DNA.

In vitro system for middle T4 RNA. II. Studies with T4-modified RNA polymerase.

In vitro system for middle T4 RNA. II. Studies with T4-modified RNA polymerase.

In vitro system for middle T4 RNA. I. Studies with Escherichia coli RNA polymerase.

We describe crude in vitro systems from T4-infected cells which reflect in vivo T4 regulation. Lysates from cells which had been infected in the presence of chloramphenicol manifest the same polarity of RNA synthesis as did the infected cells. Next, we describe a complementation system between lysates which have no RNA synthetic capacity and purified RNA polymerase; in this system, delayed early RNA synthesis in vitro depends on the presence of an active mot gene product. Mot activity controls middle mode gene expression in vivo. In vitro, not activity in the lysate directs RNA polymerase to initiate on regions of DNA that are otherwise inaccessible. This mot-dependent delayed early RNA synthesis in vitro is seen at 0.1 and 0.2 M KCl, but not at 0.05 M KCl. We present a model in which mot is a DNA melting protein necessary for recognition of a middle promoters by either Escherichia coli or T4-modified RNA polymerase which contains E. coli sigma subunit.

Phage T4-modified RNA polymerase transcribes T4 late genes in vitro.

Initiation of T4 late RNA synthesis has been achieved in an in vitro system prepared from Escherichia coli cells infected with wild-type or maturation-defective mutant T4 phage. The system uses a cellophane membrane as a mechanical support for concentrated cell lysates and for added streptolydigin-resistant RNA polymerases. Transcriptional activity and selectivity of added RNA polymerases are tested while endogenous RNA polymerase activity is inhibited by streptolydigin. T4-modified RNA polymerase is required for substantial stimulation of T4 late RNA synthesis.

Purification and Properties of the NAD+: Protein ADP‐ribosyltransferase Responsible for the T4‐Phage‐Induced Modification of the œ Subunit of DNA‐Dependent RNA Polymerase of Escherichia coli

The NAD+: protein ADP‐ribosyltransferase responsible for the bacteriophage‐T4‐induced modification of RNA polymerase has been purified 100–200‐fold from Escherichia coli B/r infected with the phage T4.The enzyme has a molecular weight around 26000. It does not require magnesium ions and is protected by sulfhydryl reagents. The temperature optimum is around 20°C. The reaction is inhibited at increased ionic strength and by the reaction products nicotinamide and T4‐modified RNA polymerase. The reaction is irreversible. The only reaction products formed to a significant extent in a crude extract are T4‐modified a subunit and one smaller ADP‐ribosylated peptide. As in vivo, the reaction is complete. Both œ subunits in the RNA polymerase monomer are ADP‐ribosylated in one and the same position.The product of modification in vitro appears to be identical with that of modification in vivo. ADP‐ribose has been obtained by hydrolysis both of T4‐modified œ subunit and of the reaction product of egg‐white lysozyme, NAD+ and the transferase responsible for bacteriophage‐T4‐induced alteration of host proteins [Rohrer, H., Zillig, W., and Mailhammer, R. (1975) Eur. J. Biochem. 60, 227–238]. The amino acid carrying the ADP‐ribosyl residue appears to be arginine. T4‐modified œ subunit and the major species of T4‐altered œ subunit are identical.An NAD+: protein ADP‐ribosyltransferase using E. coli proteins and egg‐white lysozyme but no RNA polymerase subunit as acceptor(s) has been purified from uninfected E. coli cells.

The interaction bacterial and phage proteins with immobilized Escherichia coli RNA polymerase.

Abstract The interaction of Escherichia coli RNA polymerase with other proteins has been studied by affinity chromatography. Cyanogen bromide-activated agarose covalently binds RNA polymerase, and the bound protein is enzymatically active. A column containing immobilized polymerase will retain other proteins which interact with that enzyme; these proteins can be subsequently eluted and identified. The E. coli transcriptional factor σ is one such RNA polymerase-binding protein. Certain bacteriophage T4 proteins bind to polymerase-agarose, including the products of T4 maturation control genes 33 and 55. One phage T7 protein (the 0.3 protein, of unknown function) also binds to RNA polymerase. Using this convenient affinity probe, which provides a one-step analysis of crude cell extracts, I have screened related phage systems for RNA polymerase-binding proteins: T2 and T6 produce binding proteins comparable to those of T4; whereas T3 lacks a protein equivalent to the 0.3 protein of T7. Resins containing core RNA polymerase, holoenzyme (core enzyme and σ), and polymerase modified by T4 infection display different specificities for viral proteins. In particular, the T4-modified RNA polymerase column specifically retains the product of T4 DNA synthesis gene 45.