Role of T7 RNA Polymerase His784 in Start Site Selection and Initial Transcription † Luis G. Brieba, Robert Padilla, and Rui Sousa* Department of Biochemistry, UniVersity of Texas Health Sciences Center, 7703 Floyd Curl DriVe, San Antonio, Texas 78284-7760 ReceiVed December 18, 2001; ReVised Manuscript ReceiVed February 12, 2002 ABSTRACT: The role of steric constraints vs sequence preference in start site selection by T7 RNA polymerase was investigated by using a series of synthetic promoters in which the preferred template strand ‘CC’ initiation sequence was moved away from its normal position relative to the -17 to -6 element of the T7 promoter. It was found that the CC sequence directs efficient initiation if placed 1 or 2 nt downstream of its normal position, but not if placed upstream, or more than 2 nt downstream, of +1. Mutagenesis revealed that part of the bias to initiate with GTP is due to an interaction between histidine 784 and the 2-amino group of a guanosine bound in the initiating triphosphate position. This interaction is also important for holding short transcripts within the transcription complex during initial transcription. The 23 base pair T7 promoter has a tripartite structure, with distinct elements important for specific binding of T7 RNAP, 1 for promoter opening, and for facilitating the initiation and extension of transcripts to a length which allows formation of a stable elongation complex (EC). The sequence from -17 to -6 is critical for sequence-specific binding of the polymerase (1-9). Mutations in this region reduce promoter-polymerase affinity, and the crystal struc- ture of an RNAP-promoter complex reveals numerous interactions with bases in this region (10, 11). Mutations in the -4 to -1 ‘TATA’ sequence reduce promoter activity, but can be compensated for by negative supercoiling (3, 4), suggesting that the role of this element is to facilitate promoter opening. In fact, while the -17 to -6 sequence must be duplex to direct specific binding of the polymerase, the promoter can be single-stranded downstream of -5 and still be fully active (12). Crystal structures (10, 11) and permanganate reactivity (13, 14) confirm that the -1 to -4 sequence is melted during initiation, but that the upstream sequence remains duplex. The third part of the promoter is formed by the sequence immediately downstream of the transcription start site (+1 to +6; the initially transcribed sequence or ITS). The nontemplate (NT) strand ITS of the most active T7 promoters is ‘GGGAGA’ (15). Changes to this sequence reduce initiation rates, increase apparent NTP K s values, and increase dissociation of short transcripts from the transcription complex during initial transcription, with changes at +1 or +2 having the most severe effects (6, 16- 19). The ITS also appears to be important in specifying the transcription start site. Though start site selection is deter- mined primarily by steric constraintsstranscription usually begins 6 nt downstream of the -17 to -6 elementsthere is a strong preference for initiating with ‘GG’. In fact, if the T strand base at +1 is changed to G, A, or T, but C occurs at +2, then transcription will start from +2(6, 20). The effects of changes to the ITS are similar both in fully duplex promoters and in those lacking an NT strand downstream of -5, indicating that the NT strand is unlikely to play a major role in ITS function. The role of the ITS in directing efficient initiation and in stabilizing the association of short transcripts with the initial transcription complex (ITC) may involve multiple mechanisms. When starting with the canonical ‘GGGAGA’ sequence, purine-purine stacking and G-C hydrogen bonding may enhance binding of the initiating NTPs, as well as transcript -template and transcript- NTP interactions. However, such mechanisms should operate with any RNAP, but not all RNAPs display a strong preference for initiating with guanosines. Therefore, it is likely that T7 RNAP active site side chains also make base-specific interactions with either the T strand or the NTPs. To identify such interactions, we examined the structure of a T7 RNAP ITC and identified two side chains likely to make base-specific interactions important either for start site selection or for binding the transcript during initial transcription. Mutation of one of these (R425) severely reduces RNAP activity, and mutations at this position were not further characterized. However, the effects of mutations of H784 reveal that this residue makes an H-bond to the 2-amino group of the initiating guanosine triphosphate and the 3′-guanosine of the transcript. Thus, this interaction is important both for directing initiation with guanosines and for holding G-terminated short RNAs within the ITC during initial transcription. MATERIALS AND METHODS Mutant construction, enzyme expression, and purification were as described (21). Modified NTPs were from Trilink † Supported by the Welch Foundation, by NIH Grant GM 52522 (to R.S.), and by a Fulbright-CONACYT Fellowship (to L.G.B.). * To whom correspondence should be addressed. Phone: 210-567- 8782. Fax: 210-567-8778. E-mail: sousa@biochem.uthscsa.edu. 1 Abbreviations: RNAP, RNA polymerase; EC, elongation complex; ITS, initially transcribed sequence; NT, nontemplate; T, template; ITC, initial transcription complex; wt, wild type; TBE, Tris-borate-EDTA; nt, nucleotide(s). 5144 Biochemistry 2002, 41, 5144-5149 10.1021/bi016057v CCC: $22.00 © 2002 American Chemical Society Published on Web 03/23/2002