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Mutations affecting two different steps in transcription initiation at the phage lambda PRM promoter
Abstract:
The abortive initiation assay [McClure, W. R. (1980) Proc. Natl. Acad. Sci. USA 77, 5634-5638] was used to study the effects of mutations on the activity of the P(RM) promoter of phage lambda in vitro. The transcription initiation properties of four mutant promoters were compared with those of wild-type P(RM) in the presence or absence of repressor (which activates P(RM)). Two kinetic parameters were measured: k(2), the rate constant for the transition between closed and open complexes, and K(B), the equilibrium constant for the initial binding of RNA polymerase to DNA (formation of closed complexes). The primary effect of repressor on wild-type initiation was stimulation of the isomerization reaction: k(2) increased about 7-fold. Both in the presence and in the absence of repressor, prmU31 and prmE104 (changes at nucleotides -33 and -38, respectively) reduced K(B) significantly without affecting k(2), indicating that these mutations affect polymerase binding but not the formation of open complexes. In contrast, prmE37 (a change at nucleotide -14) reduced k(2) significantly without affecting K(B). A fourth mutation, prmE93 (at nucleotide -39), is phenotypically Prm(-) primarily because it causes a defect in the O(B)2 operator site and, therefore, the mutant promoter is unable to respond normally to repressor. These results are consistent with the idea that the two regions of Escherichia coli promoters in which consensus sequences have been identified, the regions at nucleotides -35 and -10, may provide information for two discrete steps in transcription initiation.
Insights
Mutations in phage lambda's P(RM) promoter affect transcription initiation. Some mutations impact RNA polymerase binding, while others affect the transition to open complexes, revealing promoter region roles.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The P(RM) promoter of phage lambda is crucial for regulating viral gene expression.
- Understanding promoter function requires dissecting the roles of specific DNA sequences in transcription initiation.
Purpose of the Study:
- To investigate the impact of specific mutations on the activity of the phage lambda P(RM) promoter.
- To elucidate the roles of different promoter regions in RNA polymerase binding and transcription initiation.
Main Methods:
- Utilized the abortive initiation assay to study promoter activity in vitro.
- Measured kinetic parameters: k(2) (isomerization rate) and K(B) (binding equilibrium).
- Compared wild-type P(RM) with four mutant promoters in the presence and absence of repressor.
Main Results:
- Repressor significantly stimulated the isomerization rate (k(2)) of the wild-type promoter.
- Mutations at nucleotides -33 and -38 (prmU31, prmE104) reduced RNA polymerase binding (K(B)) without affecting isomerization (k(2)).
- A mutation at nucleotide -14 (prmE37) reduced isomerization (k(2)) but not binding (K(B)).
- A mutation at nucleotide -39 (prmE93) disrupted repressor binding to the O(B)2 operator site.
Conclusions:
- The -35 and -10 regions of Escherichia coli promoters may independently influence distinct steps in transcription initiation.
- Specific promoter DNA sequences differentially affect RNA polymerase binding and the transition to the open complex.
- Mutations can selectively impair either polymerase binding or the isomerization step of transcription initiation.