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

Young-Soo Kwon,Changwon Kang

doi:10.1074/jbc.274.41.29149

Young-Soo Kwon, Changwon Kang

Open Access

https://doi.org/10.1074/jbc.274.41.29149

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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.

Highlights

When an effective termination signal for the bacteriophage T7 RNA polymerase transcription was identified in the human preproparathyroid hormone (PTH)1 gene [1], its peculiar features, different from the usual bacterial factor-independent
We examined the termination of phage SP6 and T7 RNA polymerases at various mutants of the unusual downstream signal in the E. coli rrnB t1 terminator and defined the elements that are essential for this type of termination
Termination of SP6 RNA Polymerase at the Terminator t1 of E. coli Operon rrnB—Intrinsic termination of the SP6 RNA polymerase transcription occurred on the terminator t1 (Fig. 1) at multiple sites (Fig. 2A, lane W)

Summary

Introduction

When an effective termination signal for the bacteriophage T7 RNA polymerase transcription was identified in the human preproparathyroid hormone (PTH) gene [1], its peculiar features, different from the usual bacterial factor-independent. T7 termination at two upstream sites required the formation of stable secondary structure in transcripts and did not need a nontemplate strand DNA [5]. This phenomenon was observed with E. coli RNA polymerase [6]. The rrnB t1 downstream termination signal shares a common sequence (ATCTGTT in the non-template strand) with the coding region of human PTH gene, vesicular stomatitis virus (VSV) DNA, and the concatemer junction (CJ) of the replicating T7 DNA [7], which were reported to cause pausing or termination by T7 RNA polymerase without formation of RNA secondary structure. The two types are different in their requirement for the RNA hairpin structure formation and in their recognition of a nicked form of T7 RNA polymerase

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