Effects of saturation mutagenesis of the phage SP6 promoter on transcription activity, presented by activity logos

Abstract

A full set of SP6 promoter variants with all possible single substitutions at positions ؊17 to ؉5 was constructed. Transcription activities of these variants were individually measured in vivo and in vitro to determine the contribution of each base pair to the promoter activity. The in vivo activity was measured indirectly by transcriptional interference of the replication of promoter-bearing plasmids. This activity depends most highly on residues ؊11, ؊9, ؊8, ؊7, and ؉1 (initiation site). All substitutions at ؊11, ؊9, ؊8, and ؊7 abolished formation of closed complexes, except for A؊8C. These residues are involved in base-specific interactions with the polymerase, and the substitutions exhibit the same strong inhibition in vitro. In contrast, the in vitro activities of some other variants, measured on linearized templates, were different from those in vivo. Some variants at ؊13, ؊4, and ؊2, among others, showed exceptionally higher activities in vivo than in vitro, supporting the possibility that these residues are involved in postbinding steps, including template melting and bending. The A؊3T variant showed much lower activity in vivo than in vitro, but it bound to the polymerase 2-fold more than the consensus sequence and is possibly involved in polymerase binding. A quantitative hierarchy of all the base pairs is graphically displayed by activity logos, revealing the energetic contribution of each base pair to the activity. The bacteriophage SP6 genome encodes a single-subunit RNA polymerase that is very similar to the T7, T3, and K11 RNA polymerases. SP6 promoters consist of a highly conserved 20-bp sequence that extends from positions Ϫ17 to ϩ3 and exhibits a strong homology to the T7, T3, and K11 promoter sequences (1). Despite their homology, each polymerase shows highly stringent specificity for its own promoter sequence. Mutational studies with phage T7 promoters have yielded detailed information about the relationship of structure to function and the recognition of them by RNA polymerase. The promoter consists of two domains: an initiation domain downstream of Ϫ4 and a binding domain upstream of Ϫ5 (2, 3). Single base changes in the initiation domain have little effect on promoter binding but reduce the rate of transcription initiation. In contrast, changes in the binding domain reduce the efficiency of promoter binding but have little effect on the initiation of transcription. Base pairs at positions Ϫ11 through Ϫ7 of the T7 promoter are essential to the binding (4-7).The interactions of phage RNA polymerases with their promoters have also been studied by a variety of biochemical methods. Footprinting studies with methidiumpropyl-EDTAFe(II) indicate that the T7 polymerase protects the region from Ϫ17 to Ϫ4 (8, 9). Methylation and ethylation interference studies indicate that the major groove of the promoter between Ϫ5 and Ϫ12 is important for polymerase binding (10). More recent studies with base analog substitutions reveal that, at positions Ϫ11 and Ϫ10 of the T3 and T7 promoters, their RNA polymeras...

Complete Genomic Sequence of the Virulent Salmonella Bacteriophage SP6

“…The SP6 genome was searched for sequences that correspond to the SP6RP core promoter sequence (5Ј-ATTTAGGTGACACTATAG [53]). Thirteen related sequences were identified that have three or fewer mismatches; a degenerate consensus sequence, KAWTTARG KGACACTATAG, was derived based on the promoter sequences with two or fewer mismatches; and the search was repeated.…”

“…No additional putative promoters were identified. Of the 13 sequences, 3 (ϩ1 positions at bp 2121, 6304, and 29410) are unlikely to function since they depart from the core sequence at positions 1, Ϫ1, and Ϫ15, respectively, which would dramatically decrease promoter activity (53). The remaining 10 sites are excellent candidates for SP6 promoters; based on saturation mutagenesis experiments, the departures of these sequences from the core consensus sequence should have little effect on promoter activity (Fig.…”

“…The remaining 10 sites are excellent candidates for SP6 promoters; based on saturation mutagenesis experiments, the departures of these sequences from the core consensus sequence should have little effect on promoter activity (Fig. 3) (53). The sequence with the greatest departure from the core consensus is P3, which has substitutions at positions Ϫ12, Ϫ16, and Ϫ17, although mutational studies (53) indicated that these substitutions do not have a large impact on promoter activity (Fig.…”

The Genome of Bacteriophage K1F, a T7-Like Phage That Has Acquired the Ability To Replicate on K1 Strains of Escherichia coli

“…This exlusivity of promoter specificity seems to be the trend among the T7-like phages (24,42,45), and it seems that there must be some selective pressure that resulted in this feature.…”

Identification of Bacteriophage K11 Genomic Promoters for K11 RNA Polymerase

“…1). Our previous study of saturation mutagenesis on the SP6 promoter suggested that the residue at −10 hardly contributes to transcription activity in vivo or in vitro (Shin et al, 2000), although it is mostly T among the 11 known SP6 promoters. Also, the SP6 residues at −12 are highly diverse and contribute little to promoter activity.…”