We have characterized an unusual type of termination signal for T7 RNA polymerase that requires a conserved 7-base pair sequence in the DNA (ATCTGTT in the nontemplate strand). Each of the nucleotides within this sequence is critical for function, as any substitutions abolish termination. The primary site of termination occurs 7 nucleotides downstream from this sequence but is context-independent (that is, the sequence around the site of termination, and in particular the nucleotide at the site of termination, need not be conserved). Termination requires the presence of the conserved sequence and its complement in duplex DNA and is abolished or diminished if the signal is placed downstream of regions in which the non-template strand is missing or mismatched. Under the latter conditions, much of the RNA product remains associated with the template. The latter results suggest that proper resolution of the transcription bubble at its trailing edge and/or displacement of the RNA product are required for termination at this class of signal.A variety of signals have been found to modulate the process of transcript elongation. In general, these have been categorized as falling into the following three classes: pause sites, which temporarily halt the RNA polymerase (RNAP) 1 but subsequently allow resumption of transcription; termination signals, which cause release of the RNA and dissociation of the transcription complex; and arrest sites, at which the RNAP may be halted for a prolonged period but may escape by cleavage and subsequent elongation of the transcript (for review, see Refs. 1 and 2). Among the termination signals, the best characterized involve the formation of a stem-loop structure in the nascent RNA (3-5). Although there have been reports of pause, arrest, or termination signals that do not involve the formation of a structured RNA (see for example Ref. 6), these signals have been less well studied. In this work, we have characterized a sequence-specific pause/termination signal for T7 RNAP and have identified the elements that are required for its function.Two types of signals are known to cause pausing and/or termination by T7 RNAP (7,8). Class I terminators, typified by the signal that is present in the late region of T7 DNA (T⌽), encode RNAs that have the potential to form stable stem-loop structures followed by a run of U residues. These features are reminiscent of many intrinsic terminators utilized by Escherichia coli RNA polymerase, and a number of bacterial termination signals have been shown to cause T7 RNAP to terminate (8 -13). Although the members of this class encode RNAs that share a typical secondary structure, they exhibit little sequence homology.A second type of termination signal recognized by T7 RNAP was first identified in the cloned human prepro-parathyroid hormone (PTH) gene (8,14). These signals (class II signals) do not encode RNAs with an apparent consistent secondary structure but share a common sequence (ATCTGTT, in the nontemplate strand (8,15,16); this work). Additional membe...

While the binding region of the T7 promoter must be double-stranded (ds) to function, the non-template strand in the initiation region is dispensable, and a promoter that lacks this element allows efficient initiation. To determine whether the binding region serves merely to recruit the RNA polymerase (RNAP) to the vicinity of a melted initiation region or provides other functions, we utilized a GAL4-T7 RNAP fusion protein to provide an independent binding capacity to the RNAP. When the GAL4-T7 RNAP was recruited to a single-stranded (ss) promoter via a nearby Gal4 recognition sequence, no transcription was observed. However, transcription from the ss promoter could be activated by the addition, in trans, of a ds hairpin loop that contains only the binding region of the promoter. The same results were obtained in the absence of the GAL4 recognition sequence in the template and were also observed with wild type enzyme. Gel-shift experiments indicate that exposure of the RNAP to the isolated binding region facilitates recruitment of the ss template, but that the binding region is displaced from the complex prior to initiation. We conclude that exposure of the RNAP to the isolated binding region reorganizes the enzyme, allowing it to bind to the ss template. These findings have potential implications with regard to mechanisms of promoter binding and melting.To initiate transcription, RNA polymerases (RNAP) 1 must locate and bind to a promoter region, separate the two DNA stands near the start site, and position the template strand in the active center. Understanding how this process occurs is important to our knowledge of the transcription process. As is the situation with other RNAPs, promoters for T7 RNA polymerase have a bipartite structure that consists of an upstream binding region that is recognized in a sequence-specific manner and an initiation region that must be melted open prior to initiation (1). The binding region (Ϫ17 to Ϫ5) appears to function independently of the initiation region, and a double-stranded (ds) DNA fragment that contains only this region of the promoter is tightly bound by the RNAP (2). The binding region is recognized only in the form of duplex DNA, and removal of either the template (T) or non-template (NT) strands in this region prevents polymerase binding (3). In contrast, the NT strand in the initiation region is not required for initiation, and a partially singlestranded (pss) promoter in which this strand has been removed downstream of Ϫ4 (essentially a "premelted promoter") allows efficient and accurate initiation (3).A variety of lines of evidence suggests that the binding and initiation regions function independently and that it may be possible to physically separate the two while retaining promoter function. In the crystal structure of a T7 RNAP-promoter complex, as well as in an initiation complex in which the first 3 bases in the template strand have been transcribed, the two promoter domains are observed to interact with separate regions of the RNAP (4, 5). Furthermore, a...