Regulation of promoter selection by the bacteriophage T7 RNA polymerase in vitro

Abstract

During bacteriophage T7 infection a phage-specified RNA polymerase transcribes the late phage genes in two temporal classes (class II and class III). In this report, we show that the purified phage polymerase discriminates between the class II and class III promoters in vitro as a function of variables that alter the stability of the DNA helix. These variables include ionic strength, temperature, and the presence of denaturing agents such as dimethyl sulfoxide. In general, initiation at the class II promoters is preferentially inhibited as helix stability is increased. Conditions required for the establishment of salt-resistant transcription complexes by the T7 RNA polymerase have been determined; the establishment of stable complexes at the class II promoters requires the synthesis of a longer nascent RNA transcript than does formation of such complexes at the class III promoters. A comparison of the nucleotide sequences of several class II and class III promoters suggests certain features that may be responsible for the different responses of these promoters to helix destabilization. The conservation of structural features that are peculiar to the class II or class III promoters indicates that these features are important in regulation of T7 transcription in vivo. Experiments which bear on the physiological significance of these features are discussed.

Mechanism for De Novo RNA Synthesis and Initiating Nucleotide Specificity by T7 RNA Polymerase

“…T7 RNAP uses ATP as the initiating nucleotide at two natural promoters on the T7 genome. The øOL and ø2.5 promoters are, however, rather weak, [14][15][16] observations that are consistent with reduced binding of ATP in the de novo initiation complex. The ternary complexes with T-AA and T-GG showed no density for 3′-dUTP or 3′-dCTP, suggesting that their dissociation constants may be lower than the concentration (5 mM) used for crystallographic studies.…”

Extended Upstream A-T Sequence Increases T7 Promoter Strength

“…Thus, the DNA sequence in the extended upstream AT-rich region can play a role in modulating the efficiency of transcription initiation by affecting the affinity of T7 RNAP for the promoter DNA and by affecting the efficiency of promoter clearance. Thus, in addition to the promoter binding region contributing to the differences in promoter strength among class II and between class II and class III promoters, the upstream AT-rich region also appears to be involved in promoter discrimination (15,16).…”

“…In contrast, the sequence of this region is variable among the weaker class II promoters, with one or more GC bp interrupting the AT run (14). By observing the salt-regulated transcriptional activity of T7 RNAP in vitro and the usage of promoters in vivo, McAllister and colleagues (15,16) found that shortening this AT-rich region from 9 to 4 bp but keeping the integrity within the Ϫ13 to Ϫ17 region was sufficient to make a class III promoter act more like a weaker class II promoter. Promoter 3.8 is the only class II promoter with uninterrupted AT runs from Ϫ13 to Ϫ22, and it behaves more like a class III promoter (14,15).…”

Expression of HSP86 in male germ cells.

“…Within the seminiferous tubules, the physical organization of the germ cells reflects the extent of differentiation, so that the least mature stem cells are located furthest outside, and the most mature spermatazoa are located inside the lumen (15,16). To determine which cell types in the testis express HSP86, the distribution of HSP86 mRNA was analyzed by in situ hybridization (20) with antisense and control sense RNAs transcribed in vitro (8,13). The antisense HSP86 RNA hybridized in a ringlike pattern to the outside portion of the seminiferous tubules, corresponding to the distribution of immature spermatogenic cells (Fig.…”