Amino-terminal sequences of sigma N (sigma 54) inhibit RNA polymerase isomerization

Abstract

In bacteria, association of the specialized N protein with the core RNA polymerase subunits forms a holoenzyme able to bind promoter DNA, but unable to melt DNA and initiate transcription unless acted on by an activator protein. The conserved amino-terminal 50 amino acids of N (Region I) are required for the response to activators. We have used pre-melted DNA templates, in which the template strand is unpaired and accessible for transcription initiation, to mimic a naturally melted promoter and explore the function of Region I. Our results indicate that one activity of Region I sequences is to inhibit productive interaction of holoenzyme with pre-melted DNA. On pre-melted DNA targets, either activation of N -holoenzyme or removal of Region I allowed efficient formation of complexes in which melted DNA was sequestered by RNA polymerase. Like natural pre-initiation complexes formed on conventional DNA templates through the action of activator, such complexes were heparin-resistant and transcriptionally active. The inhibitory N Region I domain functioned in trans to confer heparin sensitivity to complexes between Region I-deleted holoenzyme and pre-melted promoter DNA. Evidence that Region I senses the conformation of the promoter was obtained from protein footprint experiments. We suggest that one function for Region I is to mask a single-strand DNA-binding activity of the holoenzyme. On the basis of extended DNA footprints of Region I-deleted holoenzyme, we also propose that Region I prevents RNA polymerase isomerization, a conformational change necessary for access to and the subsequent stable association of holoenzyme with melted DNA.

Roles for the C-terminal Region of Sigma 54 in Transcriptional Silencing and DNA Binding

“…The linkage was proposed to occur through the Ϫ12 recognition element because certain promoter mutations there lead to bypass transcription (14,15). Mutants with greater effects on isomerization than on binding have been reported (9,19,21,38,39), and some of the mutants identified here may fall in that category. Some of these are within the N terminus where there is at least one cluster of residues with a role in mediating activation (33,36).…”

“…The structure would promote both the maintenance of the silenced state and the changes that occur at the fork with activation. The changes that accompany activation include an unmasking of the determinants that strengthen binding to downstream single-stranded DNA (6,9,11,38). All N-terminal bypass mutations studied thus far (9,38) 2 display this enhanced downstream binding, indicating that their effects extend beyond simply interfering with binding the silencing fork structure.…”

“…The changes that accompany activation include an unmasking of the determinants that strengthen binding to downstream single-stranded DNA (6,9,11,38). All N-terminal bypass mutations studied thus far (9,38) 2 display this enhanced downstream binding, indicating that their effects extend beyond simply interfering with binding the silencing fork structure. The two C-terminal mutants are shown here to behave in a simpler manner; they retain downstream binding, but they do not strengthen it.…”

Cyclic Di-AMP Homeostasis in Bacillus subtilis

“…Based on this finding, it is tempting to speculate that the N-terminal helices are involved in the control of the activity of the DAC domain and thus in the sporespecific synthesis of c-di-AMP. Similarly, inhibitory protein domains have been found in the B. subtilis transcription factor RocR and the alternative factor 54 (51,52). Future studies will focus on the distinct molecular mechanisms controlling the c-di-AMP levels in B. subtilis.…”

Investigating protein–protein interfaces in bacterial transcription complexes: a fragmentation approach

“…Activator-dependent transcription initiation by the s 54 -RNAP requires the amino terminal region 1 of s 54 . (25,26) In the absence of region 1, the roadblock normally associated with s 54 -RNAP transcription initiation is overcome and closed complexes can, in vitro, isomerise unaided to form open complexes, inferring that region 1 is the target for the activator. (27) Using a truncated form of s 54 (region 1 deleted, DR1s 54 ), it was observed that the amount of ternary complex formed between DR1s 54 bound to the earlymelted probe and the PspF fragment was significantly reduced, consistent with direct binding of region 1 to the PspF: ADP-AlFx complex.…”