Stimulation of the phage λ pL promoter by integration host factor requires the carboxy terminus of the α-subunit of RNA polymerase

Giladi, Hilla; Igarashi, Kazuhiko; Ishihama, Akira; Oppenheim, Amos B.

doi:10.1016/0022-2836(92)90514-k

Cited by 54 publications

(44 citation statements)

References 34 publications

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“…Subsequent studies demonstrated that mutant RNA polymerases containing these truncated ao subunits were impaired in activation by OmpR (31), A CII (25), Pseudomonas TrpI (25), Ada (53), and OxyR (62). Stimulation of the A PL promoter by integration host factor was also abolished (22). In the cases of CAP at lacP1 (38) and OxyR at katG (62), loss of cooperative binding of the activator and RNA polymerase was associated with the truncated a subunits.…”

Section: Discussionmentioning

confidence: 99%

Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein

et al. 1994

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The bacteriophage P2 ogr gene product is a positive regulator of transcription from P2 late promoters. The ogr gene was originally defined by compensatory mutations that overcame the block to P2 growth imposed by a host mutation, rpoA109, in the gene encoding the a subunit of RNA polymerase. DNA sequence analysis has confirmed that this mutation affects the C-terminal region of the a subunit, changing a leucine residue at position 290 to a histidine (rpoAL290H). We have employed a reporter plasmid system to screen other, previously described, rpoA mutants for effects on activation of a P2 late promoter and have identified a second allele, rpoA155, that blocks P2 late transcription. This mutation lies just upstream of rpoAL290H, changing the leucine residue at position 289 to a phenylalanine (rpoAL289F). The effect of the rpoAL289F mutation is not suppressed by the rpoAL29OH-compensatory P2 ogr mutation. P2 ogr mutants that overcome the block imposed by rpoAL289F were isolated and characterized. Our results are consistent with a direct interaction between Ogr and the a subunit of RNA polymerase and support a model in which transcription factor contact sites within the C terminus of a are discrete and tightly clustered.Many eubacterial genes and operons are under positive control, and the structures and DNA-binding sites of a number of prokaryotic transcriptional activators have been well characterized (for a review, see reference 1). The precise mechanism by which these regulatory proteins catalyze the initiation of transcription remains largely obscure. The identification of lambda repressor (9, 24, 27, 28) and catabolite gene activator protein (CAP) (3, 15, 65) mutants that were defective as activators but retained DNA-binding ability suggested that these proteins function by interacting directly with RNA polymerase. A rapidly growing body of genetic evidence supports a functional interaction between the C-terminal region of the ax subunit(s) of DNA-dependent RNA polymerase and specific transcriptional activators.The earliest indication that the a subunit plays a role in positive control was provided by Escherichia coli mutation rpoA109 (61), which prevents lytic growth of phage P2 and satellite phage P4 by interfering with phage late-gene expression. This mutation results in a leucine-to-histidine change in the C-terminal region of the ax subunit (16 general pattern that has emerged from the characterization of these mutants is that they are altered in the C-terminal region of the a polypeptide and that each rpoA mutation appears to be fairly specific, affecting only one or a small group of positively regulated promoters. Those mutations that affect the responsiveness of RNA polymerase to each activator tend to be tightly clustered, suggesting that each transcriptional activator interacts with a discrete domain within the C-terminal region of the a subunit to stimulate transcription.To further define a domain of the ao subunit involved in interaction with the activators of P2 late-gene expression, we have dete...

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Section: Discussionmentioning

confidence: 99%

Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein

et al. 1994

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“…domain of ␣ is required for IHF-mediated stimulation of transcription at p L (Giladi et al, 1992b), the rpoA341 mutation does not appear to impair the interaction between IHF and the ␣ subunit). As synthesis and decay of the N antiterminator protein also appears to be normal in phageinfected rpoA341 bacteria, the decreased level of longer transcripts derived from the p L and p R promoters must result from inefficient N-mediated antitermination.…”

Section: Figmentioning

confidence: 93%

An RNA polymerase α subunit mutant impairs N‐dependent transcriptional antiterminationin Escherichia coli

Obuchowski

Węgrzyn

Szalewska-Pałasz

et al. 1997

Molecular Microbiology

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SummaryWe show that the rpoA341 mutation in the gene encoding the ␣ subunit of Escherichia coli RNA polymerase results in a decreased level of transcripts originating from the lytic promoters p L and p R of infecting phage. However, using lacZ fusions we demonstrate that initiation of transcription from both p L and p R is not impaired in the rpoA341 host. Rather, it is the level of the longer, antiterminated p L -and p R -derived transcripts which is altered: the activity of ␤-galactosidase in bacteria harbouring a source of N and a p L -nutL-t L1 -t I -lacZ or p R -nutR-t R1 -lacZ fusion is considerably lower in the rpoA341 mutant relative to the rpoA + strain. In the absence of the antiterminator protein N no difference is observed in the level of longer p R -derived transcripts between wild-type (rpoA + ) and mutant (rpoA341) hosts. Although synthesis of N appears to be similar in both phageinfected rpoA + and rpoA341 cells, overexpression of the N gene leads to restoration of wild-type levels of the longer p L -and p R -derived transcripts in the mutant host. While this mutation does not appear to affect vegetative phage growth in nus + backgrounds, in combination with certain nus mutations it retards lytic development. Therefore, we conclude that the rpoA341 mutation specifically interferes with the function of the N-antitermination complex, suggesting that the C-terminal domain of the RNA polymerase ␣ subunit may play an important role in N-dependent transcriptional antitermination.

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“…For both the pL promoter and the Mu Pe promoter, it has been shown that the presence of the ␣CTD is required for direct activation (14,43). The results described here show that the same amino acid (R-265) that is involved in binding of the ␣CTD to DNA is also important for the IHF-mediated activation.…”

Section: Discussionmentioning

confidence: 57%

“…In these promoters, the ihf site is located around position Ϫ85, or Ϫ95 in the upstream Pe promoter region. Since transcription from the pL1 and Mu Pe promoters could no longer be stimulated by IHF in vitro, when the ␣CTD was deleted (14,43), IHF was classified as a class I activator. In this paper, we searched for amino acid residues in the ␣CTD involved in IHF-mediated activation of the Pe promoter, by using the same set of alanine substitutions used for identification of the targets of the CRP (37) and the UP element (12).…”

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confidence: 99%

Function of the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase in basal expression and integration host factor-mediated activation of the early promoter of bacteriophage Mu

et al. 1997

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Transcription initiation in Escherichia coli is influenced by promoter architecture and by the presence of regulatory proteins, which can either stimulate or repress this process (for a review, see reference 6). A classical E. coli promoter contains the Ϫ35 and Ϫ10 elements; both the sequence and the spacing of these elements are important determinants for promoter strength. Sequences upstream of the Ϫ35 element can increase promoter activity (2, 5). It was shown in the rrnB P1 promoter that sequences between Ϫ40 and Ϫ60 (the so-called UP element) increase transcription by interacting with the alpha subunit of RNA polymerase (12, 33). Mutant RNA polymerase, lacking the C-terminal domain of the alpha subunit (␣CTD), was unable to contact the UP element, and subsequently, transcription from the rrnB P1 promoter was less efficiently initiated (33). The ␣CTD is a distinct domain of 64 amino acids which can dimerize and bind the DNA (3). The structure of this domain has been solved by nuclear magnetic resonance (21). By using the alanine scan method, in which every amino acid of two regions of the ␣CTD one by one was exchanged for an alanine, the residues at positions 265, 268, 269, 296, 298, and 299 were identified as important for UP-element binding (12).A number of bacterial activators which generally bind to a site close to the promoter and which stimulate the initiation of transcription have been identified (20). Of these activators, the cyclic AMP receptor protein (CRP) is the best studied (reviewed in reference 23). Two classes of promoters stimulated by CRP can be distinguished (20). For class I promoters, with the CRP binding site located upstream of the Ϫ35 element, most frequently around position Ϫ61, contacts have been found between the activator and the alpha subunit of the RNA polymerase complex. Mutation and cross-linking studies revealed that the target for CRP interaction also resides in the ␣CTD (7,47,48). An alanine scan identified glutamic acid 261 as one of the most important residues for this contact (37). In class II promoters, the CRP binding site is located around position Ϫ41 where it overlaps with the classical Ϫ35 promoter element. Although CRP bound at such a site makes contact with the alpha subunit, mutant RNA polymerase lacking the ␣CTD is still activated by CRP (1,45,47).

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Stimulation of the phage λ pL promoter by integration host factor requires the carboxy terminus of the α-subunit of RNA polymerase

Cited by 54 publications

References 34 publications

Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein

Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein

An RNA polymerase α subunit mutant impairs N‐dependent transcriptional antiterminationin Escherichia coli

Function of the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase in basal expression and integration host factor-mediated activation of the early promoter of bacteriophage Mu

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