Promoter Efficiencies Can Be Increased or Decreased by Mutation

KEY CONCEPTS
- Down mutations to decrease promoter efficiency usually decrease conformance to the consensus sequences, whereas up mutations have the opposite effect.
- Mutations in the −35 sequence can affect initial binding of RNA polymerase.
- Mutations in the −10 sequence can affect binding or the melting reaction that converts a closed to an open complex.
Effects of mutations can provide information about promoter function. Mutations in promoters affect the level of expression of the gene(s) they control without altering the gene products themselves. Most are identified as bacterial mutants that have lost, or have very much reduced, transcription of the adjacent genes. They are known as down mutations. Mutants are also found with up mutations in which there is increased transcription from the promoter.
It is important to remember that “up” and “down” mutations are defined relative to the usual efficiency with which a particular promoter functions. This varies widely. Thus a change that is
recognized as a down mutation in one promoter might never have been isolated in another (which in its wild-type state could be even less efficient than the mutant form of the first promoter).
Information gained from studies in vivo simply identifies the overall direction of the change caused by mutation. Mutations that increase the similarity of the −10 or −35 elements to the consensus sequences or bring the distance between them closer to 17 bp usually increase promoter activity. Likewise, mutations that decrease the resemblance of either site to the consensus or make the distance between them farther from 17 bp result in decreased promoter activity. Down mutations tend to be concentrated in the most highly conserved promoter positions, confirming the particular importance of these bases as determinants of promoter efficiency. However, exceptions to these rules occasionally occur.
For example, a promoter with consensus sequences in all the modules . However, no such natural promoters exist in the E. coli genome, and artificial promoters with “perfect” matches to the consensus at all these positions are actually weaker than promoters with at least one mismatch in the −10 or −35 consensus hexamers. This is because they bind to RNA polymerase so tightly that this actually impedes promoter escape.
To determine the absolute effects of promoter mutations, the affinity of RNA polymerase for wild-type and mutant promoters has been measured in vitro. Variation in the rate at which RNA polymerase binds to different promoters in vitro correlates well with the frequencies of transcription when their genes are expressed in vivo. Taking this analysis further, the stage at which a mutation influences the efficiency of a promoter can be determined.
Does it change the affinity of the promoter for binding RNA polymerase? Does it leave the enzyme able to bind but unable to initiate? Is the influence of an ancillary factor altered? By measuring the kinetic constants for formation of a closed complex and its conversion to an open complex, we can dissect the two stages of the initiation reaction:
- Down mutations in the −35 sequence usually reduce the rate of closed complex formation, but they do not inhibit the conversion to an open complex.
- Down mutations in the −10 sequence can reduce either the initial formation of a closed complex or its conversion to the open form, or both.
The consensus sequence of the −10 site consists exclusively of A-T base pairs, a configuration that assists the initial melting of DNA into single strands. The lower energy needed to disrupt A-T pairs compared with G-C pairs means that a stretch of A-T pairs demands the minimum amount of energy for strand separation. The sequences immediately around and downstream from the start point also influence the initiation event. Furthermore, the initial transcribed region (from about +1 to about +120) influences therate at whic h RNA polymerase clears the promoter, and therefore has an effect upon promoter strength. Thus, the overall strength of a promoter cannot always be predicted from its consensus sequences, even when taking into consideration the other RNA polymerase recognition elements in addition to the −10 and −35
elements.
It is important to emphasize that although similarity to consensus is a useful tool for identifying promoters by DNA sequence alone, and “typical” promoters contain easily recognized −35 and −10 sequences, many promoters lack recognizable −10 and/or −35 elements. In many of these cases, the promoter cannot be recognized by RNA polymerase alone and requires an ancillary protein “activator”  that overcomes the deficiency in intrinsic interaction between RNA polymerase and the promoter. It is also important to emphasize that “optimal activity” does not mean “maximal activity.” Many promoters have evolved with sequences far from consensus precisely because it is not optimal for the cell to make too much of the product encoded by the RNA transcript.

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