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Figure 7 DNA-crosslinking to PspF1–275 is depende...

Figure 3. PspF1–275 F85Y is the only F85 variant i...

Figure 7. Crosslinking profiles of PspF1–275·σ54·D...

Figure 5. PspF1–275 is similarly organized in all ...

Fig. 5 The activities of the V132A and L138A va...

Figure 1. The cryo-electron microscopy structure o...

Figure 6. Determinants in PspF1–275 required for f...

Fig. 4 Gel filtration profiles of PspF1−275 (WT...

Figure 4. PspF1–275:ADP–BeF alters the σ54–DNA int...

Figure 2. PspF1–275:ADP–BeF supports stable trappe...

Figure 4.: σ54 variants which cannot bind PspF1–275 normally in the presence of ADP·AlFx. ADP·AlFx dependent complexes formed between σ54 and PspF1–275 were detected by Coomassie staining. σ54 and PspF1–275 were presented at 1 and 20 μM separately. Arrow (a) indicates the complex formed between σ54 and PspF1–275 in the presence of ADP·AlFx. Arrow (b) indicates the changed position of PspF1–275 complex in the presence of ADP·AlFx. Arrow (c) and (d) indicates the new bands formed in this assay.

Image Text (High Precision): 1-275 111 PspF

Other Images from "Construction and functional analyses of a comprehensive σ54 site-directed mutant library using alanine–cysteine mutagenesis":


Figure 5. (A) S. meliloti nifH heteroduplex promot...

Figure 7. The left panel shows the cryo-EM reconst...

Figure 3. Formation of holoenzyme by σ54 variants ...

Figure 4. σ54 variants which cannot bind PspF1–275...

Figure 2. In vivo stability of σ54 variants which ...

Figure 6. Mutations in σ54 that affect transcripti...

Figure 1. Diagram of the functional regions of σ54...

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Abstract

The σ54 factor associates with core RNA polymerase (RNAP) to form a holoenzyme that is unable to initiate transcription unless acted on by an activator protein. σ54 is closely involved in many steps of activator-dependent transcription, such as core RNAP binding, promoter recognition, activator interaction and open complex formation. To systematically define σ54 residues that contribute to each of these functions and to generate a resource for site specific protein labeling, a complete mutant library of σ54 was constructed by alanine–cysteine scanning mutagenesis. Amino acid residues from 3 to 476 of Cys(-)σ54 were systematically mutated to alanine and cysteine in groups of two adjacent residues at a time. The influences of each substitution pair upon the functions of σ54 were analyzed in vivo and in vitro and the functions of many residues were revealed for the first time. Increased σ54 isomerization activity seldom corresponded with an increased transcription activity of the holoenzyme, suggesting the steps after σ54 isomerization, likely to be changes in core RNAP structure, are also strictly regulated or rate limiting to open complex formation. A linkage between core RNAP-binding activity and activator responsiveness indicates that the σ54-core RNAP interface changes upon activation.


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