The bacteriophage T4 late-transcription coactivator gp33 binds the flap domain of Escherichia coli RNA polymerase

Nechaev et al. 10.1073/pnas.0408028101.

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Fig. 6. Competition between AsiA and gp33 for binding s ⁷⁰ holoenzyme. A mixture containing 5 pmol of s ⁷⁰ and, as required, 40 pmol of AsiA was combined with 1 pmol of RNAP core in a final volume of 4.5 m l. After a 5-min incubation at 25°C, reactions were supplemented with increasing quantities of [³²P]gp33 and incubated for an additional 5 min at 25°C. One microliter of each reaction mixture was loaded on a 4-15% native PHAST gel, and gels were processed as described in Materials and Methods. (A) Effect of AsiA on gp33 binding to wild-type s ⁷⁰ holoenzyme. The graph represents binding of gp33 to s ⁷⁰ holoenzyme in the absence or presence of AsiA as a function of the gp33:RNAP core ratio. s ^70D 3,4-holoenzyme served as a positive control. (B) Comparison of the effects of AsiA on gp33 binding to wild-type s ⁷⁰ holoenzyme and s ⁷⁰(R541C/L607P)-holoenzyme.

Fig. 7. Peptide scanning of T4 gp33. A membrane containing a set of 15-mer synthetic peptides spanning the entire gp33 sequence, in 2 aa steps, was probed for RNAP binding, as described in Materials and Methods. Each spot indicated by a dot corresponds to an individual peptide. T4 gp33 peptides yielding positive signals are marked P1-P3. Sequence and the predicted secondary structure of T4 gp33 amino acids 30-92 are shown on top (shading and secondary structure assignments are as in Fig. 4), with P1-P3 peptides indicated by horizontal lines.

Fig. 8. Repression of basal T4 late transcription by wild-type and mutant gp33. (A) RNAP core was combined with gp55 and various quantities of wild-type or mutant gp33. After a 15-min incubation at 25°C, a single round of transcription was initiated by simultaneous addition of rNTPs and heparin. Reaction products were processed as described in Materials and Methods. T, transcript; RM, recovery marker. (B) Quantitative analysis. Transcription by wild-type (WT) and D b 900-909 RNAPs in the absence of gp33 was normalized to 1.

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