Relative efficiency of utilization of promoter and termination sites by bacteriophage T3 RNA polymerase

Bacteriophage T3 RNA polymerase promoters have been classified as class II and class III on the basis of their relative location in T3 DNA as well as on the function of the protein products encoded by the messages transcribed from them. In the present work, the efficiency of utilization of several class II and class III promoters by bacteriophage T3 RNA polymerase was compared with regard to (a) rate of initiation of transcription as determined by [³²P]PP(i) exchange with GTP; (b) complex formation between polymerase and promoters in the presence of GTP; and (c) competition between different promoters for T3 RNA polymerase in a standard transcription assay. The results of these experiments indicated that the class II promoters at 1.05 and 22.8 T3 map units, whose promoter sequences are remarkably similar to the consensus class III promoter sequences, are nearly as strong as typical class III promoters. In contrast, the class II promoter at 14.3 T3 map units, whose promoter sequence differs from the consensus class III promoter sequence by having a C:G base pair instead of a usual A:T base pair at the -1 position, was considerably weaker than the class III promoter. When the C:G base pair at this position was changed to A:T using site-directed mutagenesis, the rate of initiation of RNA synthesis from the mutant promoter was similar to that of a typical class III promoter. In agreement with this observation, it was observed that changing the A:T base pair at the -1 position of a strong class II promoter, at 1.05 T3 map units, to C:G decreased the rate of RNA synthesis from this promoter by about 65%. These observations indicate that the nucleotide residues at the -1 position play a critical role in determining the efficiency of promoter utilization by T3 RNA polymerase. The two termination sites recognized in vitro by bacteriophage T3 RNA polymerase on the T3 genome have been cloned, sequenced, and mapped. Analysis of the DNA nucleotide sequence surrounding the termination site at 59.7 map units indicated that the putative RNA transcript arising from this region can be arranged into a GC-rich stem-loop structure followed by a U-rich 3' tail. However, a major fraction of T3 RNA polymerase molecules read through this terminator in vitro to transcribe regions of T3 DNA beyond this terminator. In contrast to termination at 59.7 map units, termination of transcription at 100 T3 map units does not occur in response to any putative terminator structure or sequence. Rather, T3 RNA polymerase transcribes through the terminal repeat region to the end of T3 DNA molecules to yield run-off transcripts.