DNA-Dependent Synthesis of RNA:- RNA Synthesis Begins at Promoters
Initiation of RNA synthesis at random points in a DNA molecule would be an extraordinarily wasteful process. Instead, an RNA polymerase binds to specific sequences in the DNA called promoters, which direct the transcription of adjacent segments of DNA (genes). The sequences where RNA polymerases bind can be quite variable, and much research has focused on identifying the particular sequences that are critical to promoter function.
In E. coli, RNA polymerase binding occurs within a region stretching from about 70 bp before the transcription start site to about 30 bp beyond it. By convention, the DNA base pairs that correspond to the beginning of an RNA molecule are given positive numbers, and those preceding the RNA start site are given negative numbers. The promoter region thus extends between positions 70 and 30. Analyses and comparisons of the most common class of bacterial promoters (those recognized by an RNA polymerase holoenzyme containing 70) have revealed similarities in two short sequences centered about positions 10 and 35 (Fig. 2). These sequences are important interaction sites for the δ70 subunit. Although the sequences are not identical for all bacterial promoters in this class, certain nucleotides that are particularly common at each position form a consensus sequence (recall the E. coli oriC consensus sequence). The consensus sequence at the 10 region is (5) TATAAT (3); the consensus sequence at the 35 region is (5) TTGACA (3). A third AT-rich recognition element, called the UP (upstream promoter) element, occurs between positions 40 and 60 in the promoters of certain highly expressed genes. The UP element is bound by the subunit of RNA polymerase. The efficiency with which an RNA polymerase binds to a promoter and initiates transcription is determined in large measure by these sequences, the spacing between them, and their distance from the transcription start site.
Many independent lines of evidence attest to the functional importance of the sequences in the 35 and 10 regions. Mutations that affect the function of a given promoter often involve a base pair in these regions. Variations in the consensus sequence also affect the efficiency of RNA polymerase binding and transcription initiation. A change in only one base pair can decrease the rate of binding by several orders of magnitude. The promoter sequence thus establishes a basal level of expression that can vary greatly from one E. coli gene to the next. A method that provides information about the interaction between RNA polymerase and promoters is illustrated in Box 26–1.
The pathway of transcription initiation is becoming much better defined (Fig. 3a). It consists of two major parts, binding and initiation, each with multiple steps. First, the polymerase binds to the promoter, forming, in succession, a closed complex (in which the bound DNA is intact) and an open complex (in which the bound DNA is intact and partially unwound near the 10 sequence). Second, transcription is initiated within the complex, leading to a conformational change that converts the complex to the elongation form, followed by movement of the transcription complex away from the promoter (promoter clearance). Any of these steps can be affected by the specific makeup of the promoter sequences. The subunit dissociates as the polymerase enters the elongation phase of transcription (Fig. 3a). E. coli has other classes of promoters, bound by RNA polymerase holoenzymes with different subunits. An example is the promoters of the heat-shock genes. The products of this set of genes are made at higher levels when the cell has received an insult, such as a sudden increase in temperature. RNA polymerase binds to the promoters of these genes only when δ70 is replaced with the δ32 (Mr 32,000) subunit, which is specific for the heat-shock promoters (see Fig. 1). By using different δ subunits the cell can coordinate the expression of sets of genes, permitting major changes in cell physiology.
FIGURE 2 Structure of the RNA polymerase holoenzyme of the bacterium Thermus aquaticus. (Derived from PDB ID 1IW7.) 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 β subunit is in gray, the subunit is white; the two subunits are different shades of red; the ω subunit is yellow; the δ 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 ω subunit is visible.
FIGURE 3 Typical E. coli promoters recognized by an RNA polymerase holoenzyme containing 70. 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 ring in the region between -40 and -60, strongly stimulate transcription at the promoters that contain them. The UP element in the rrnB P1 promoter encompasses the region between -38 and -59. The consensus sequence for E. coli promoters recognized by δ70 is shown second from the top. Spacer regions contain slightly variable numbers of nucleotides (N). Only the first nucleotide coding the RNA transcript (at position +1) is shown.
