T4 Middle Promoters Research Articles

An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription.

The bacteriophage T4 MotA protein is a transcriptional activator of T4-modified host RNA polymerase and is required for activation of the middle class of T4 promoters. MotA alone binds to the -30 region of T4 middle promoters, a region that contains the MotA box consensus sequence [(t/a)(t/a)TGCTT(t/c)A]. We report the isolation and characterization of a protein designated Mot21, in which the first 8 codons of the wild-type motA sequence have been replaced with 11 different codons. In gel retardation assays, Mot21 and MotA bind DNA containing the T4 middle promoter P(uvsX) similarly, and the proteins yield similar footprints on P(uvsX). However, Mot21 is severely defective in the activation of transcription. On native protein gels, a new protein species is seen after incubation of the sigma70 subunit of RNA polymerase and wild-type MotA protein, suggesting a direct protein-protein contact between MotA and sigma70. Mot21 fails to form this complex, suggesting that this interaction is necessary for transcriptional activation and that the Mot21 defect arises because Mot21 cannot form this contact like the wild-type activator.

Characterization of Pre-transcription Complexes Made at a Bacteriophage T4 Middle Promoter: Involvement of the T4 MotA Activator and the T4 AsiA Protein, a σ 70Binding Protein, in the Formation of the Open Complex

Bacteriophage T4 middle promoters have excellent matches to the −10 consensus sequence for the σ 70subunit of Escherichia coliRNA polymerase, but a binding site for the T4 transcriptional activator MotA replaces the σ 70−35 consensus. E. coliRNA polymerase transcribes from middle promoters with or without the activator. In contrast, transcription by T4-modified E. coliRNA polymerase, which is present during T4 infection, requires MotA. We show that transcription by unmodified polymerase from the T4 middle promoter P uvsXis independent of the specific sequences within the −35 region, and the DNase I footprint obtained with unmodified polymerase and P uvsXresembles those seen previously with E. coli"extended −10" promoters. In contrast, although T4-modified polymerase alone binds P uvsX, promoter unwinding and detection of a DNase I footprint requires MotA. This footprint is significantly different from that obtained with unmodified polymerase, starting upstream of around position −20. Previous work has indicated that the T4 AsiA protein, which binds tightly to σ 70, is the phage modification required for MotA activation. We show that in the presence of AsiA, MotA, and otherwise unmodified polymerase, DNase I protection of P uvsXis now similar to that obtained with the fully modified polymerase and MotA up to around position −40. However, protection upstream of −40 is still similar to that seen with unmodified polymerase. Our results support the idea that MotA-dependent activation requires AsiA binding to σ 70to achieve specific protein-DNA contacts within the −20 to −40 region of a middle promoter.

Bacteriophage T4 middle transcription system: T4-modified RNA polymerase; AsiA, a σ70 binding protein; and transcriptional activator MotA

This chapter discusses the purification of the components of the middle transcription system, T4-modified host RNA polymerase, MotA protein, and Asia protein; and the MotA-dependent transcription reaction from middle promoters in vitro. An in vitro transcription system is developed for studying MotA-dependent transcription from T4 middle promoters. Using partially purified RNA polymerase isolated after T4 infection and highly purified MotA protein, transcription from several T4 middle promoters has been demonstrated in vitro. Further characterization and purification of the fully modified polymerase will allow the identification of any other T4 functions that are involved in MotA-dependent transcription from middle promoters. The level of MotA-dependent transcription from PuvsX by the fully modified polymerase is dependent on the reaction conditions. Reproducibly high levels of MotA-dependent transcription are observed when using the 1× Kglu buffer and the incubation conditions given earlier. However, the use of very high chloride concentrations, such as the 1× KCl transcription buffer, results in much less transcription as the level of MotA to the DNA is decreased. In addition, the pretranscription complex made in a buffer with a high chloride concentration is less stable to heparin challenge than that made in the glutamate buffer. It is not clear whether this phenomenon is related to the decreased activity of the T4-modified polymerase seen in the general assay or to the chloride sensitivity noted for the phage-modified polymerase in very early work.

Bacteriophage T4 MotA and AsiA proteins suffice to direct Escherichia coli RNA polymerase to initiate transcription at T4 middle promoters.

Development of bacteriophage T4 in Escherichia coli requires the sequential recognition of three classes of promoters: early, middle, and late. Recognition of middle promoters is known to require the motA gene product, a protein that binds specifically to the "Mot box" located at the -30 region of these promoters. In vivo, the asiA gene product is as critical for middle mode RNA synthesis as is that of the motA gene. In vitro, AsiA protein is known to loosen the sigma 70-core RNA polymerase interactions and to inhibit some sigma 70-dependent transcription, presumably through binding to the sigma 70 subunit. Here we show that, in vitro, purified MotA and AsiA proteins are both necessary and sufficient to activate transcription initiation at T4 middle promoters by the E. coli RNA polymerase in a sigma 70-dependent manner. AsiA is also shown to inhibit recognition of T4 early promoters and may play a pivotal role in the recognition of all three classes of phage promoters.

The bacteriophage T4 middle promoter PuvsX: analysis of regions important for binding of the T4 transcriptional activator MotA and for activation of transcription.

Bacteriophage T4 middle promoters, which are transcribed using phage-modified host RNA polymerase and the T4 transcriptional activator, MotA, match the host sigma 70 consensus sequence at -10, but they have a different consensus ((t/a)(t/a)TGCTT(t/c)A) (a MotA box) at -30. While the T4 middle promoter PuvsX has these -10 and -30 motifs, it also has matches to the MotA box at -35, -51, -70, and -87. We show that MotA binds to PuvsX DNA, footprinting a region that includes the MotA boxes at -30, -35, and -51. Very high levels of MotA are required for footprinting and gel-shift experiments, and protein-DNA complexes formed in the presence of both phage-modified polymerase and MotA are more resistant to HindIII cleavage than those formed with either protein alone. These results suggest that MotA-DNA interactions may be stabilized by phage-modified polymerase. Sequences between -18 and -38 are absolutely required for MotA activation of transcription, but sequences upstream of -38 are stimulatory, particularly when chloride instead of glutamate is the major anion. Our results dissect PuvsX into a core promoter, downstream of -38, which is required for MotA activation, and an upstream region that enhances transcription especially under conditions less favourable for protein-DNA interactions.

The asiA gene product of bacteriophage T4 is required for middle mode RNA synthesis

The asiA gene of bacteriophage T4 encodes a 10-kDa peptide which binds strongly in vitro to the sigma 70 subunit of Escherichia coli RNA polymerase, thereby weakening sigma 70-core interactions and inhibiting sigma 70-dependent transcription. To assess the physiological role of this protein, we have introduced an amber mutation into the proximal portion of the asiA gene. On suppressor-deficient hosts, this mutant phage (amS22) produces minute plaques and exhibits a pronounced delay in phage production. During these mutant infections, T4 DNA synthesis is strongly delayed, suggesting that the AsiA protein plays an important role during the prereplicative period of phage T4 development. The kinetics of protein synthesis show clearly that while T4 early proteins are synthesized normally, those expressed primarily via the middle mode exhibit a marked inhibition. In fact, the pattern of protein synthesis after amS22 infection resembles greatly that seen after infection by amG1, an amber mutant in motA, a T4 gene whose product is known to control middle mode RNA synthesis. The amber mutations in the motA and asiA genes complement, both for phage growth and for normal kinetics of middle mode protein synthesis. Furthermore, primer extension analyses show that three different MotA-dependent T4 middle promoters are not recognized after infection by the asiA mutant phage. Thus, in conjunction with the MotA protein, the AsiA protein is required for transcription activation at T4 middle mode promoters.

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.

Transcriptional mapping of a DNA replication gene cluster in bacteriophage T4. Sites for initiation, termination, and mRNA processing.

A phage T4 genetic cluster that encodes DNA polymerase, several other DNA replication proteins, transcriptional factors, and the translation repressor RegA has been shown to be controlled by overlapping modes of transcription which initiate at several promoters. The promoters were mapped by using a combination of assays including Northern blotting, S1-mapping, RNA sequencing, and analysis of products of radioactive labeling of 5' ends on T4-induced RNA in vitro via the reaction catalyzed by eukaryotic guanylyl transferase (RNA capping assay). The most proximal in the cluster are two promoters that do not require any phage-induced factors for activation, i.e. they are T4 early promoters. Initiation at these promoters yields several RNA species having overlapping 5'-terminal sequences, the largest of which is estimated to be about 15,000 nucleotides long and to include all the cistrons of the cluster. A third early promoter maps inside the protein encoding segment of one of the cistrons (T4 gene 47), while at least five additional promoters map in intercistronic regions and are T4 middle promoters, i.e. they require the T4-induced DNA-binding transcription factor MotA. Transcriptional readthrough at a termination site within the T4 gene 45-44 intercistronic region is required for synthesis of gp44 and gp62, two essential T4 DNA-polymerase (gp43) accessory proteins. In contrast, transcription of T4 gene 43 is serviced by readthrough across a termination site in the regA-43 intercistronic region as well as by a MotA-dependent promoter that maps downstream of the termination site, and the region contains a site for processing by a T4-induced enzyme that also cleaves elsewhere in the polycistronic mRNA from the cluster (i.e. in the Shine-Dalgarno sequence of the gene 45.2 mRNA). The termination events in the gene 45-44 and regA-43 intercistronic regions both occur downstream of RNA stem-loop structures containing the sequence 5'CUUCGG3' in the loop segments. Transcription termination in the 78-base-pair regA-43 intercistronic region occurs about 60 nucleotides away from the gp43 initiator AUG, transcription initiation occurs at 38-40 nucleotides upstream from the AUG, and T4-dependent RNA processing occurs at several sites (including a GGAG sequence) between the transcription termination and initiation sites. Thus, all gp43-encoding mRNAs contain the translational operator (residues -40 to -1 relative to the AUG) for autogenous repression by this DNA polymerase (Andrake et al., 1988).(ABSTRACT TRUNCATED AT 400 WORDS)

Transcriptional activation of bacteriophage T4 middle promoters by the motA protein

Transcriptional activation of middle genes in bacteriophage T4 requires the phage-encoded motA protein. Many middle genes are involved in deoxyribonucleotide biosynthesis and phage DNA replication. In the absence of motA, the gene products that are required for DNA synthesis are transcribed from other, upstream promoters. Using primer extension sequencing on RNA templates isolated from T4 motA + and motA − infected cells, we have characterized 14 motA-dependent transcripts. The T4 middle promoters have a consensus sequence of nine base-pairs, ( ca t )( a t )TGCTT( t c )A, spaced 11 to 13 nucleotides away from the Escherichia coli −10 consensus sequence, TAnnnT. The motA protein also can act as a transcriptional repressor for at least one early gene. Furthermore, the phage-encoded motA protein can activate in trans a middle promoter resident on a plasmid.