RNA polymerase Holoenzyme

GTFs and RNA polymerase with promoters—or in one step by the recognition of the promoter by a preformed GTF-RNA polymerase holoenzyme complex. 

Marr, M.T., Datwyler, S.A., Meares, C.F., and Roberts, J.W. (2001) Restructuring of an RNA polymerase holoenzyme elongation complex by lambdoid phage Q proteins. PNAS 98, 8972-8978. 

RapA RapA E. coli RNA polymerase core or RNA polymerase holoenzyme Recycling of RNA polymerase [332]. 

Transcription initiation in procaryotes is controlled via promoters and regulatory DNA sequences located near the promoter. The role of the promoter is to provide a defined association site for the RNA polymerase and to correctly orient it. The binding of the RNA polymerase to its promoter is controlled by the sigma factor, a component of the RNA polymerase holoenzyme. The sigma factor selects which genes are to be transcribed by specifically recognizing the promoter sequence and structure and by allowing the RNA polymerase to form a transcription-competent complex at the transcription start site. 

This type of promoter displays markedly different characteristics compared to the o -dependent promoter. The o -contammg holoenzyme binds tightly to the promoter in the absence of transcriptional activators. In this closed state, however, it is not capable of initiating transcription. The transcriptional activators are required in this case to activate the promoter-bound holoenzyme for initiation, i.e. to transform it into the open complex (see Fig. 1.29). Activation is mediated via protein-protein interactions between the transcriptional activator and the RNA polymerase holoenzyme, and is accompanied by ATP hydrolysis. The binding site for the transcriptional activator is found at a distance of ca.llO bp upstream form the start site and can be shifted further upstream without loss of stimulatory effect. Direct interaction of the holoenzyme with the bound transcriptional activator is possible due to loop formation of the intervening DNA. The strict dependency on transcriptional activators for transcription initiation indicates that the DNA-bound holoenzyme alone is not capable of isomerizing to the transcription-competent open complex. The transition to the open complex requires interactions with the transcriptional activator, an event which occurs with ATP hydrolysis. 

Fig. 1.30. Structure of a typical eucaryotic transcription start site. Enhancer elements and UAS elements (UAS upstream activating sequences) are binding sites for positive and negative regulatory DNA-binding proteins. The TATA box is the binding site for the TATA box binding protein (TBP) and serves to position the RNA polymerase holoenzyme on the promoter. For promoters that do not possess a TATA box, this function is fulfilled by an initiator region.

FIGURE 26-4 Structure of the RNA polymerase holoenzyme of the bacterium Thermus aquaticus. (Derived from PDB ID 1 IW7.)The overall structure of this enzyme is very similar to that of the E. coli RNA polymerase no DNA or RNA is shown here. The j3 subunit is in gray, the j3 subunit is white the two a subunits are different shades of red the to subunit is yellow the cr subunit is orange. The image on the left is oriented as in Figure 26-6. When the structure is rotated 180° about the y axis (right) the small to subunit is visible. 

FIGURE 26-5 Typical E. coli promoters recognized by an RNA polymerase holoenzyme containing a70. Sequences of the nontemplate strand are shown, read in the 5 —>3 direction, as is the convention for representations of this kind. The sequences vary from one promoter to the next, but comparisons of many promoters reveal similarities, particularly in the —10 and -35 regions. The sequence element UP, not present in all E. coli promoters, is shown in the P1 promoter for the highly expressed rRNA gene rrnB. UP elements, generally occur-... 

Sequences within the prokaryotic — promoter region that are recognized by the RNA polymerase holoenzyme... 

Figure 29.5 RNA polymerase holoenzyme complex. Notice that the a subunit (orange) of the bacterial RNA polymerase holoenzyme makes sequence-specific contacts with the -10 and - 5 promoter sequences (yellow). [From K. S. Murakami, S. Masuda. E. A. Campbell. 0. Muzzin, and S, A. Darst. Science 296 2002) 1285-1290.]...

Key elements of dependent promotors are the TATA box, with the consensus sequence TATAAT 10 bp upstream from the transcription initiation site (pos. -10), and the sequence TTGACA at the position -35 (Fig. 1.18). Both sequences are necessary for the recognition of the promotor by a70. Structural analysis of the Thermus aquaticus RNA polymerase holoenzyme bound to DNA shows that all sequence-specific contacts with the core promotor are mediated by the sigma subunit (Murakami et al. 2002). This archaeal RNA polymerase has a subunit structure (a ji/i oxr) similar to that of the eubacterial enzyme. The intervening sequences, as well as other upstream sequences, can also influence the efficiency of transcription initiation. It is not possible to define consensus sequences at these positions. An optimal dependent promotor can be defined as a sequence with the - 35 hexamer as well as the -10 hexamer 17 bp away. The latter lies 7 bp upstream from the transcription initiation site. 

Mediators are a class of proteins that mediate the contacts between specific transcription factors and the basal transcription complex. These proteins are found as part of RNA polymerase holoenzyme forms. 

Murakami, K.S., Masuda, S., Campbell, E.A., Muzzin, O. and Darst, S.A. (2002) Structural basis of transcription initiation an RNA polymerase holoenzyme-DNA complex. Science, 296, 1285-1290. 

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