Initiation

Initiation comprises all the steps necessary to begin RNA synthesis, including (1) promoter recognition, (2) formation of a transcription bubble, (3) creation of the first bonds between rNTPs, and (4) escape of the transcription apparatus from the promoter.

Transcription initiation requires that the transcription apparatus recognize and bind to the promoter. At this step, the selectivity of transcription is enforced: the binding of RNA polymerase to the promoter determines which parts of the DNA template are to be transcribed, and how often. Different genes are transcribed with different frequencies, and promoter binding is primarily responsible for determining the frequency of transcription for a particular gene. Promoters also have different affinities for RNA polymerase. Even within a single promoter, the affinity can vary with the passage of time, depending on the promoter’s interaction with RNA polymerase and a number of other factors.

BACTERIAL PROMOTERS Promoters, as we have seen, are DNA sequences that are recognized by the transcription apparatus and are required for transcription to take place. Essential information for the transcription apparatus—where it will start transcribing, which strand is to be read, and in what direction the RNA polymerase will move—is embedded in the nucleotide sequence of the promoter. In bacterial cells, promoters are usually adjacent to an RNA-coding sequence.

An examination of many promoters in E. coli and other bacteria reveals a general feature: although most promoters vary in sequence, short stretches of nucleotides are common to many. Furthermore, the spacing and location of these nucleotides relative to the transcription start site are similar in most promoters. These short stretches of common nucleotides are called consensus sequences because they possess considerable similarity, or consensus (Figure 10.9). The presence of consensus in a set of nucleotides usually implies that the sequence is associated with an important function. image TRY PROBLEM 26

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Figure 10.9: A consensus sequence consists of the most commonly encountered bases at each position in a group of related sequences.

The most commonly encountered consensus sequence, found in almost all bacterial promoters, is centered about 10 bp upstream of the start site. Called the −10 consensus sequence, or sometimes the Pribnow box, this consensus sequence,

5′—TATAAT—3′

3′—ATATTA—5′

is often written simply as TATAAT (Figure 10.10). Remember that TATAAT is just the consensus sequence—­representing the most commonly encountered nucleotides at each of these positions. In most prokaryotic promoters, the actual sequence is not TATAAT.

Another consensus sequence common to most bacterial promoters is TTGACA, which lies approximately 35 nucleotides upstream of the start site and is termed the 35 consensus sequence (see Figure 10.10). The nucleotides on either side of the −10 and −35 consensus sequences and those between them vary greatly from promoter to promoter, suggesting that they are not very important in promoter recognition.

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Figure 10.10: In bacterial promoters, consensus sequences are found upstream of the start site, approximately at positions −10 and −35.

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The sigma factor associates with the core RNA polymerase (Figure 10.11a) to form a holoenzyme, which binds to the −35 and −10 consensus sequences in the DNA promoter (Figure 10.11b). The holoenzyme initially binds weakly to the promoter, but then undergoes a change in structure that allows it to bind more tightly and unwind the double-stranded DNA (Figure 10.11c). The holoenzyme extends from −50 to +20 when bound to the promoter. Unwinding begins within the −10 consensus sequence and extends downstream for about 14 nucleotides, including the start site (from nucleotides −12 to +2).

CONCEPTS

A promoter is a DNA sequence, usually adjacent to a gene, that is required for gene transcription. Promoters contain short consensus sequences that are important in the initiation of transcription.

INITIAL RNA SYNTHESIS After the holoenzyme has attached to the promoter, RNA polymerase is positioned over the start site for transcription (at position +1) and has unwound the DNA to produce a single-stranded template. The orientation and spacing of consensus sequences on a DNA strand determine which strand will be the template for transcription and thereby determine the direction of transcription.

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The position of the start site is determined not by the sequences located there, but by the location of the consensus sequences, which position RNA polymerase so that the enzyme’s active site is aligned for the initiation of transcription at +1. If the consensus sequences are artificially moved upstream or downstream, the location of the starting point of transcription correspondingly changes.

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Figure 10.11: Transcription in bacteria is catalyzed by RNA polymerase, which must bind to the sigma factor to initiate transcription.

To begin the synthesis of an RNA molecule, RNA polymerase pairs the base on a ribonucleoside triphosphate with its complementary base at the start site on the DNA template strand (Figure 10.11d). No primer is required to initiate the synthesis of the 5′ end of the RNA molecule. Two of the three phosphate groups are cleaved from the ribonucleoside triphosphate as the nucleotide is added to the 3′ end of the growing RNA molecule. However, because the 5′ end of the first ribonucleoside triphosphate does not take part in the formation of a phosphodiester bond, all three of its phosphate groups remain. An RNA molecule therefore possesses, at least initially, three phosphate groups at its 5′ end (Figure 10.11e).