Open complex formation by Escherichia coli RNA polymerase: the mechanism of polymerase‐induced strand separation of double helical DNA

Abstract

SummaryEscherichia coli RNA polymerase is able to sitespecifically melt 12bp of promoter DNA at temperatures far below those normally associated with DNA melting. Here we consider several models to explain how RNA potymerase destabilizes duplex DNA. One popular model proposes that upon binding to the promoter, RNA polymerase untwists the spacer DNA between the 10 and 35 regions, which results in a destabilization of the 10 region at a TA base step where melting initiates. Promoter untwisting may result, in part, from extensive wrapping of the DNA around RNA polymerase. Formation of the strandseparated open complex appears to be facilitated by specific protein-DNA interactions which occur predominantly on the non-template strand. Recent evidence suggests that these include important contacts with Sigma factor region 2.3. which we propose binds the displaced single strand of DNA.

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

“…The 70 subunit plays a key role in recognizing and making specific contacts with base pairs at the Ϫ35 and Ϫ10 regions of the promoter (3). In the open complex, DNA at the Ϫ10 region presumably becomes singlestranded for RNA polymerase to start reading the exposed template strand and polymerizing ribonucleotides (4)(5)(6)(7)(8)(9)(10). The bases in the nontemplate strand make specific interactions with amino acid residues of 70 (11).…”

A mutant spacer sequence between -35 and -10 elements makes the P lac promoter hyperactive and cAMP receptor protein-independent

“…The Ϫ35 element also helps RNA polymerase binding through interaction with region 4.2 of 70 (2,5,17). It is believed that the spacer region between Ϫ35 and Ϫ10 (optimal length 17 bp) does not have any specific sequence requirement and simply facilitates the spatial alignment of the Ϫ10 and Ϫ35 elements in binding to the 2.4 and 4.2 regions of the factor (7,17,18). The second kind, called ''extended Ϫ10'' promoters, contains an extra 2-bp 5Ј-TG-3Ј sequence located 1 bp 5Ј to the Ϫ10 element, (consensus sequence Ϫ15 TGNTATAAT Ϫ7 ) (19).…”

“…Both kinds require a 6-bp Ϫ10 sequence (consensus 5Ј-TATAAT-3Ј) located Ϸ7 bp 5Ј to the transcription start site. Functionally, the Ϫ10 element participates in RNA polymerase binding by interacting with the region 2.3-2.4 of 70 (2)(3)(4)(5)(6)(7)(8)(9)(10) and is part of an Ϸ15-nt putative single-stranded region in the open complex (4,8,(11)(12)(13)(14)(15). The first kind of promoters (Ϫ35 promoters) is more common and characterized by the presence of the Ϫ10 element as well as a 6-bp (consensus sequence 5Ј-TTGACA-3Ј) in the Ϫ35 position (16).…”

Supportive and inhibitory elements of a putative PrfA‐dependent promoter inListeria monocytogenes

“…Although the new sequence could still allow the formation of a possible stem-loop structure (Fig. 5C-F), this putative structure would be probably located outside of the initiation loop (deHaseth and Helmann, 1995;Craig et al, 1998). The in vitro transcription results obtained (Fig.…”

“…This putative secondary structure does not lead to transcription termination as in vitro transcription starting at ParoA1 only 15 bp upstream of this anticipated stem-loop structure is not affected by this sequence. We rather suggest that because of the formation of this secondary structure in the melted singlestranded transcription initiation bubble (deHaseth and Helmann, 1995;Craig et al, 1998) the transcriptional start nucleotide may become inaccessible for the RNA polymerase.…”