| Figures |
 |
Figure 1 The effect of gp33 on T4 late open promoter complexes. (A) Open promoter complexes monitored by KMnO4 probing. RNAP was combined with gp33, where indicated (Step 1), followed by the addition of P23 promoter DNA, 32P-labeled on the transcribed (template) strand and incubation for 5 or 20 min, as specified. Alternatively, gp33 was added to preformed open promoter complexes (Step 2). DNA cleavage at T +2, -6, and -8, indicative of promoter opening, is shown by asterisks. (B) Open complexes monitored by DNase I footprinting. Complexes containing the indicated RNAP were formed for 20 min, with gp33 added before DNA (Step 1) or after open complex formation (Step 2), as indicated. Heparin was added (to 100  g/ml) for 30 s prior to DNase addition. Lanes A: A-sequence ladders; lanes 1: DNA without protein. The DNA segment protected by the open promoter complex is indicated by the vertical bar, with gp33-dependent changes highlighted by striping.
|
 | Figure 2 Gp33 prevents RNAP binding to internal DNA sites, but not to DNA ends. (A) Repression of transcription by gp33. Open complexes were formed on a 125 bp template containing a T4 late P23 promoter; where indicated, gp33 was added before DNA (Step 1) or after the formation of the open promoter complex (Step 2). Transcripts originating at the promoter and DNA ends are identified as P and E, respectively, and their predicted lengths are indicated in brackets. P1 and E1 are run-off transcripts; longer transcripts are products of template switching. RM: labelled DNA recovery marker. (B) The effect of gp33 on nonspecific DNA binding, monitored by DNase I footprinting. The same 125 bp P23 DNA was labelled in the nontranscribed (lanes 1–6) or transcribed (lanes 7–12) strands. RNAP core or gp55 holoenzyme was incubated with DNA for 20 min at 25°C and treated with DNase I in the absence of heparin, except for samples for lanes 5 and 11, to which heparin was added 30 s prior to DNase. Where indicated, gp33 was added to RNAP prior to DNA. The extent of DNA end that is protected by RNAP in the presence of gp33 is shown by brackets.
|
 |
Figure 3 Transcription by  70 holoenzyme can be repressed by gp33. (A) The sequences of the 126 bp consensus extended -10 (-10 Econ), and 125 bp T4 late (P23) promoter templates differ only within the box-enclosed segment. Only the nontranscribed strands are shown. (B) Single-round transcription on the -10 extended consensus promoter template with wild-type RNAP core and the indicated 70-derivative holoenzymes was performed as for Figure 2A. The bottom panel shows P1 transcripts in a shorter exposure, to emphasize the 1 bp transcript shortening referred to in the text. Transcripts indicated by (x), obtained with wild-type 70 holoenzyme, are of unknown origin.
|
 | Figure 4 Repression of transcription of heteroduplex DNA by gp33. Top: Single rounds of transcription of 117 bp P23-based heteroduplex templates, with the extent of unpairing indicated above the gel. Gp33 was added to free RNAP for reactions shown in the even-numbered lanes. Leftward (L) and rightward (R) transcripts are identified at the right. Bottom: sequence of the P23 promoter template; nucleotide numbering is relative to the start of the R transcript; the T4 late promoter is written in lower case; the region changed to create mismatches is underlined; initiating nucleotides of the L and R transcripts are in bold and are marked with arrows.
|
 |
Figure 5 Nonspecific DNA binding by 70 RNAP holoenzyme derivatives. (A) Comparison of DNase I footprints of gp55- and 70-RNAP holoenzymes binding DNA probes containing cognate or noncognate promoters in the absence of heparin (except for lane 9). Complexes were assembled on 260 bp DNA (extended upstream by 136 bp relative to the standard  125 bp DNA templates) containing a consensus extended -10 ('-10 Econ') (lanes A and 1–5) or T4 late ('P23') promoter (lanes 6–11), 5' end-labelled on the nontranscribed strand. Lane A: an A sequence ladder; lanes 1 and 6: digestion patterns for DNA. 70mut is the R541C/L607P double mutant. DNA segments protected by RNAP in open promoter complexes are indicated. (B) DNA-end binding by 70 1.1 holoenzyme, monitored on 125 bp P23 promoter DNA labelled in the nontemplate strand. The extent of DNA end protected by RNAP is indicated. (C) Transcription of the 126 bp extended -10 consensus promoter template by RNAP core (lane 1), wild-type 70 (lane 2), and 701.1 (lane 3) holoenzymes. Transcripts originating at the promoter (P) and DNA ends (E) are indicated as in Figure 2A.
|
 | Figure 6 The role of the upstream interaction in initiation of transcription. Binding of RNAP core and various holoenzymes to double-stranded DNA and DNA ends (panel C) is schematized. (+) marks complexes that can bind DNA, (-) marks complexes unable to bind DNA. The horizontal double lines show DNA; white circles symbolize nonspecific DNA-binding sites on RNAP core (RNAP core itself is not shown). U and D designate, respectively, upstream and downstream interactions with DNA that are available to the specified RNAP. Grey rectangles represent promoter-specificity () subunits, with their promoter motifs and the cognate promoter motifs on DNA shown in the same color: red for gp55, green for 70, blue for 70 modified by AsiA and MotA, and black for sequence-nonspecific binding. Solid vertical lines denote RNAP–DNA interactions; blocked interactions are shown by X. Gp33 is shown in yellow; AsiA, MotA, and their cognate MotA box in DNA in blue; the grey circle in panel I symbolized the gp45 sliding clamp. (A) RNAP core binds DNA sequence nonspecifically. (B) Gp33 prevents RNAP core binding to DNA by blocking the upstream interaction. (C) The upstream interaction is dispensable for DNA end binding. (D) Wild-type 70 holoenzyme interacts with its cognate promoter through -35 and -10 promoter motifs. (E) Gp33 prevents 704.2 holoenzyme binding to DNA. (F) Changing promoter specificity by modulating the upstream interaction by the accessory proteins AsiA and MotA. Kinking of the sigma symbol represents the remodelling of 70 domain 4 that is induced by AsiA and MotA. (G) T4 late basal transcription: gp55 holoenzyme binds to DNA through two interactions, one specific and the other nonspecific; unlike 70, gp55 does not block the downstream DNA-binding site on RNAP core (symbolized by placing the gp55 symbol below the DNA). (H) Repressed T4 late transcription: gp33 blocks the nonspecific upstream interaction. (I) Activated T4 late transcription: DNA-loaded gp45 restores the upstream interaction; tethering of the sliding clamp in the promoter is secured by interactions with the C-termini of gp55 and gp33 that are not indicated.
|
|
|
