Bipartite Modular Structure of Intrinsic, RNA Hairpin-independent Termination Signal for Phage RNA Polymerases

Abstract

The phage SP6 RNA and T7 RNA polymerases, which are closely related to each other, intrinsically stop at two signals in the Escherichia coli rrnB terminator t1 through different mechanisms. The downstream signal functioned without an RNA secondary structure formation, in which the signal was still active when separated from the upstream, hairpin-forming signal, and IMP incorporation enhanced its efficiency. The sequence from -15 to -1 was essential for the downstream, hairpin-independent termination (at -1). The results of SP6 transcription with heteroduplex templates and ribonucleotide analogs suggested that the downstream signal consists of two functionally different modules. The effects of iodo-CMP or IMP incorporation into RNA on termination efficiency were not sensitive to incorporation at -9 and upstream, but they were reactive to incorporation at -6 and -2, as reflected by strong iodo-rC:dG and weak rI:dC base pairing. Thus, the downstream module (from -8 approximately -6 to -1) appears to facilitate the release of RNA. Mismatches in the templates at -6 to +1 allowed for efficient termination, unlike those upstream of the sequence. The upstream module (from -15 to -9 approximately -7) functions as a duplex. Pausing of the SP6 elongation complex at the termination site was detected when RNA release was suppressed by the incorporation of 5-bromo-UMP, and it was dependent on the upstream module. Results of single-round SP6 transcriptions using 3'-deoxynucleotides and immobilized templates indicated that RNA was not released from the elongation complexes halted at the termination site on the template variants carrying mutations in the upstream or downstream module, whereas such complexes on the wild type template were dissociated. Thus, halting or simple pausing was not sufficient for termination even when the downstream module was intact. The upstream module appears to mediate such conformation change necessary for termination.