Molecular genetic analysis of a prokaryotic transcriptional coactivator: functional domains of the bacteriophage T4 gene 33 protein

Winkelman, J W; Kassavetis, George A.; Geiduschek, E. Peter

doi:10.1128/jb.176.4.1164-1171.1994

Cited by 15 publications

(8 citation statements)

References 39 publications

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“…Moreover, as already mentioned, conditions of macromolecular crowding promote the interaction between gp45 and T4 DNA polymerase (19). These parallels have led some ofus to speculate that the mechanism by which gp45 enhances transcription and the mechanism by which gp45 generates processive DNA synthesis are identical: both occur because gp45 retains weakly interacting proteins on the DNA template (6,25). Although gp45 does not provide an auxiliary upstream DNA-binding site for the E gpS5gp33 RNA polymerase in the manner of the E. coli cAMP receptor protein, CRP, (26,27), it is capable of generating a comparable effect on transcriptional initiation by effectively confining the RNA polymerase-promoter interaction to a one-dimensional space in place of a three-dimensional space.…”

Section: Resultsmentioning

confidence: 99%

Use of a macromolecular crowding agent to dissect interactions and define functions in transcriptional activation by a DNA-tracking protein: bacteriophage T4 gene 45 protein and late transcription.

Sanders

Kassavetis²,

Geiduschek³

1994

Proc. Natl. Acad. Sci. U.S.A.

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We have used a molecular crowding reagent to define functions in the tansptional activation of bacteriophage T4 late genes. This activation normally requires the three T4 DNA polymerase accessory proteins encoded by T4 genes 44, 62, and 45 (the gp44/62 complex and gp45), an enhancer-like cis-acting site, an RNA polymerase-bound coactivator, and an unobstructed path along the DNA joining the promoter to the enhancer. We show that molecular crowding eliminates the requirement for the gp44/62 complex and for the enhancer, retains the requirement for gp45 and Its coactivator, and generates activated promoter complexes with nearly canged DNase I footprints. These experiments identify gp45 as the direct activator of tription, and the gp44/62 complex as the assembly factor for gp45. They suggest that the enhancer serves as the normal, but not invariably essential, entry site for the gp45 DNA-tracking protein. (3)(4)(5)(6).A molecular model that accounts for the properties ofthese components has been proposed: the enhancer is the site at which the gp44/62 complex and gp45 bind to DNA and determines the orientation of that binding; ATP hydrolysis in the enhancer complex triggers the detachment of gp45, so that it is free to track bidirectionally along DNA; retention of the orientation that is imposed at the enhancer restricts productive contacts of gp45 to those molecules of gp33-and gp55-bearing RNA polymerase that face in the compatible direction (6). This model designates gp45 as the primary transcriptional activator, assigns an accessory assemblyfactor role to the gp44/62 complex, and designates the enhancer as the DNA entry site for gp45. The experiments that are described below analyze the consequences of perturbing the macromolecular interactions in this transcriptional activation system with a molecular crowding agent. We show that under conditions of macromolecular crowding, gp45 activates T4 late transcription in the absence of the gp44/62 complex. gp44-and gp62-independent activation of transcription dispenses with a requirement for the enhancer and for ATP hydrolysis and also eliminates the enhancer-imposed polarity of transcriptional activation. MATERIALS AND METHODSPolyethylene glycol (PEG) 8000, polyvinyl alcohol, dextran, and Ficoll were from Sigma. Solutions ofthese reagents were deionized on a mixed-bed resin (Bio-Rad AG5O1X8 D) before use. Other reagents were from standard sources. Nicked circular DNA was prepared as described (3). Linear DNA and relaxed circular DNA were prepared by digestion with Sca I restriction endonuclease and with calf thymus topoisomerase I, respectively. The relaxed state of DNA was verified by electrophoresis in agarose/ethidium bromide gels. Plasmids pDH310 and pDH82 have been described (4,5). Their transcription units, yielding 420-nt RNA, have identical sequence.RNA polymerase core was purified from uninfected Escherichia coli as described (5). gp55 was prepared as described (7), except that purified fractions were stored in 6 M guanidine hydrochloride in place ...

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Section: Resultsmentioning

confidence: 99%

Use of a macromolecular crowding agent to dissect interactions and define functions in transcriptional activation by a DNA-tracking protein: bacteriophage T4 gene 45 protein and late transcription.

Sanders

Kassavetis²,

Geiduschek³

1994

Proc. Natl. Acad. Sci. U.S.A.

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“…In vitro the protein appears to have a stimulatory effect on other σ factors as well but in vivo seems to bias holoenzyme formation towards RpoS (Gaal et al ., 2006; Typas et al ., 2007). Transcription of bacteriophage T4 late genes is also dependent on two proteins: gp55, a σ factor distantly related to the σ 70 family, and gp33 (Kassavetis and Geiduschek, 1984; Gribskov and Burgess, 1986; Williams et al ., 1989; Winkelman et al ., 1994). gp33 interacts with the β flap of RNAP (Nechaev et al ., 2004), an interaction site also contacted by region 4 in σ 70 ‐family regulators.…”

Section: Discussionmentioning

confidence: 99%

A previously unidentified σ factor and two accessory proteins regulate oxalate decarboxylase expression in Bacillus subtilis

MacLellan

Wecke

Helmann³

2008

Molecular Microbiology

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SummaryWe have investigated the function of a cell envelope stress-inducible gene, yvrI, which encodes a 22.5 kDa protein that includes a predicted s 70 region 4 domain, but lacks an apparent region 2 domain. YvrI interacts with RNA polymerase and overexpression of YvrI results in induction of OxdC, an oxalate decarboxylase maximally expressed under low-pH conditions. We have used microarray-based analyses to define the YvrI regulon. YvrI is required for the transcription of three operons (oxdC-yvrL, yvrJ and yvrI-yvrHa) each of which is preceded by a highly similar promoter sequence. Activation of these promoters requires both YvrI and the product of the second gene in the yvrI-yvrHa operon, YvrHa. YvrI and YvrHa together allow recognition of the oxdC promoter, stimulate DNA melting and activate transcription by core RNA polymerase. Together, these results suggest that YvrI is a previously unrecognized s factor in Bacillus subtilis and that the 9.5 kDa YvrHa protein acts as a required co-activator of transcription. A yvrL deletion results in the upregulation of YvrI activity suggesting that YvrL is a negative regulator of YvrI-dependent transcription, possibly functioning as an anti-s factor.

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“…In transcriptional activation, however, the interaction of the 45 protein with the RNA polymerase is mediated by the T4-encoded 33 protein (14), which apparently contacts both the 45 protein and RNA polymerase (15). Further studies by the Geidushek group indicated that the carboxyl terminus of the 33 protein is necessary for proper interactions with the 45 protein but not with the RNA polymerase (16). Not surprisingly, the carboxyl terminus of the 33 protein and the T4 DNA polymerase are very similar, especially the last 6 aa (Fig.…”

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The carboxyl terminus of the bacteriophage T4 DNA polymerase is required for holoenzyme complex formation

Berdis

Soumillion

Benkovic

1996

Proc. Natl. Acad. Sci. U.S.A.

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To further elucidate the mechanism and dynamics of bacteriophage T4 holoenzyme formation, a mutant polymerase in which the last six carboxyl-terminal amino acids are deleted, was constructed, overexpressed, and purified to homogeneity. The mutant polymerase, designated ⌬C6 exo ؊ , is identical to wild-type exo ؊ polymerase with respect to k cat , k pol , and dissociation constants for nucleotide and DNA substrate. However, unlike wild-type exo ؊ polymerase, the ⌬C6 exo ؊ polymerase is unable to interact with the 45 protein to form the stable holoenzyme. A synthetic polypeptide corresponding to the carboxyl terminus of the wild-type exo ؊ polymerase was tested as an in vitro inhibitor of bacteriophage T4 DNA replication. Surprisingly, the peptide does not directly inhibit holoenzyme complex formation by disrupting the interaction of the polymerase with the 45 protein. On the contrary, the peptide appears to disrupt the interaction of the 44͞62 protein with the 45 protein, suggesting that the 44͞62 protein and the polymerase use the same site on the 45 protein for functional interactions. Data presented are discussed in terms of a model correlating the functionality of the carboxyl terminus of the polymerase for productive interactions with the 45 protein as well as in terms of the 45 protein concomitantly interacting with the 44͞62 protein and polymerase.The bacteriophage T4 holoenzyme is a complex of the T4 DNA polymerase and 45 protein, assembled onto duplex DNA in an ATP-dependent process by the 44͞62 protein (reviewed in refs. 1 and 2). Although intimately involved in the assembly process, the 44͞62 protein does not remain part of the holoenzyme complex but instead acts catalytically (3, 4). The role of the 45 protein is to act as a sliding clamp that tethers the polymerase to the DNA primer͞template, increasing the processivity of the polymerase. The processivity factor of the T4 system is believed to encircle DNA in a manner identical to that of the ␤ subunit (5) and proliferating cell nuclear antigen (6), based upon preliminary x-ray crystallographic studies of the 45 protein (J. Kuriyan, personal communication) as well as functional similarities among the three clamps. While the dynamics of clamp opening and͞or closing are still speculative, it is believed that the 44͞62 protein clamp loader opens and closes the 45 protein clamp by an energy transduction mechanism that closely couples the energy of ATP hydrolysis to some physical force (A.J.B. and S.J.B., unpublished data). Once loaded onto DNA, the 45 protein and polymerase form a tightly associated complex that is highly stable on duplex DNA (8). Of paramount interest and importance is the mechanism and molecular contacts describing how the sliding clamp and polymerase interact to form the stable holoenzyme.Studies of processive DNA replication from various systems have revealed the qualitative importance of the carboxylterminus of the polymerase in the functional interaction with its respective accessory protein. Herpes simplex virus holoenzyme...

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Molecular genetic analysis of a prokaryotic transcriptional coactivator: functional domains of the bacteriophage T4 gene 33 protein

Cited by 15 publications

References 39 publications

Use of a macromolecular crowding agent to dissect interactions and define functions in transcriptional activation by a DNA-tracking protein: bacteriophage T4 gene 45 protein and late transcription.

Use of a macromolecular crowding agent to dissect interactions and define functions in transcriptional activation by a DNA-tracking protein: bacteriophage T4 gene 45 protein and late transcription.

A previously unidentified σ factor and two accessory proteins regulate oxalate decarboxylase expression in Bacillus subtilis

The carboxyl terminus of the bacteriophage T4 DNA polymerase is required for holoenzyme complex formation

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