K65R is a primary reverse transcriptase (RT) mutation selected in human immunodeficiency virus type 1-infected patients taking antiretroviral regimens containing tenofovir disoproxil fumarate or other nucleoside analog RT drugs. We determined the crystal structures of K65R mutant RT cross-linked to double-stranded DNA and in complexes with tenofovir diphosphate (TFV-DP) or dATP. The crystals permit substitution of TFV-DP with dATP at the dNTP-binding site. The guanidinium planes of the arginines K65R and Arg72 were stacked to form a molecular platform that restricts the conformational adaptability of both of the residues, which explains the negative effects of the K65R mutation on nucleotide incorporation and on excision. Furthermore, the guanidinium planes of K65R and Arg72 were stacked in two different rotameric conformations in TFV-DP- and dATP-bound structures that may help explain how K65R RT discriminates the drug from substrates. These K65R-mediated effects on RT structure and function help us to visualize the complex interaction with other key nucleotide RT drug resistance mutations, such as M184V, L74V, and thymidine analog resistance mutations.

The transition from initiation to elongation of the RNA polymerase (RNAP) is an important stage of transcription that often limits the production of the full-length RNA. Little is known about the RNAP transition kinetics and the steps that dictate the transition rate, because of the challenge in monitoring subpopulations of the transient and heterogeneous transcribing complexes in rapid and real time. Here, we have dissected the complete transcription initiation pathway of T7 RNAP by using kinetic modeling of RNA synthesis and by determining the initiation (IC) to elongation (EC) transition kinetics at each RNA polymerization step using single-molecule and stoppedflow FRET methods. We show that the conversion of IC to EC in T7 RNAP consensus promoter occurs only after 8-to 12-nt synthesis, and the 12-nt synthesis represents a critical juncture in the transcriptional initiation pathway when EC formation is most efficient. We show that the slow steps of transcription initiation, including DNA scrunching/ RNAP-promoter rotational changes during 5-to 8-nt synthesis, not the major conformational changes, dictate the overall rate of EC formation in T7 RNAP and represent key steps that regulate the synthesis of full-length RNA.abortive synthesis ͉ FRET ͉ rate-limiting ͉ T7 RNA polymerase ͉ transcription transition T he recruitment of RNA polymerase (RNAP) to the promoter and isomerization to a competent open complex are important regulatory steps in gene transcription (1, 2). Similarly, the transition from abortive initiation to processive elongation is an essential event of transcription that often dictates the rate at which the full-length RNA is made (3-9). During the abortive initiation phase, the RNAP maintains contacts with the promoter and catalyzes RNA polymerization by scrunching the template DNA (10-13). After a certain length of RNA is made, the RNAP switches transcription from promoter-specific to promoter-independent and processive RNA synthesis. Single-subunit and multisubunit RNAPs switch from the initiation complex (IC) to the elongation complex (EC) by undergoing specific structural rearrangements. For example, the single-subunit T7 RNAP protein undergoes major refolding of its N-terminal domain during EC transition that releases promoter contacts and results in the formation of RNA channel (14,15). The bacterial RNAP makes this transition by releasing the sigma factor from the promoter (7,(16)(17)(18)(19), and in eukaryotic RNAP II, this event is marked by the removal of several transcription factors, including TFIIB, TFIIE, TFIIF, and TFIIH (20). Although the initiation stage and the transition from IC to EC are major points of regulation that ultimately control gene expression, the rate-limiting steps governing these multistep processes and the kinetics of transition have not been elucidated.The challenge in studying the kinetic pathway of initiation and the IC-to-EC transition has been the transient and heterogeneous nature of the initially transcribing complexes. Studies of the transient heterogen...

The kinetics of promoter binding and open complex formation in bacteriophage T7 RNA polymerase was investigated using 2-aminopurine (2-AP) modified promoters. 2-AP serves as an ideal probe to measure the kinetics of open complex formation because its fluorescence is sensitive to both base-unpairing and base-unstacking and to the nature of the neighboring bases. All four 2-AP bases in the TATA box showed an increase in fluorescence with similar kinetics upon binding to the T7 RNA polymerase, indicating that the TATA sequence becomes unpaired in a concerted manner. The 2-AP at ؊4 showed a peculiarly large increase in fluorescence upon binding to the T7 RNA polymerase. Based on the recent crystal structure of the T7 RNA polymerase-DNA complex, we propose that the large fluorescence increase is due to unstacking of the 2-AP base at ؊4 from the guanine at The bacteriophage RNA polymerases, such as the T7 RNA polymerase, are single subunit enzymes capable of catalyzing all the processes such as initiation, elongation, and termination of transcription. The structural simplicity and the template specificity of the phage RNA polymerases make them attractive for exploring the mechanism of transcription and for understanding transcription regulation at the level of protein-DNA interactions. Various crystal structures of T7 RNA polymerase as a complex with the promoter DNA and initiating nucleotides are available, and these inspire detailed studies to understand the dynamics of the transcription initiation and elongation mechanisms (1, 2).The initiation of transcription is a multistep process that directs the polymerase to the promoter region, where RNA synthesis is initiated. After the RNA polymerase recognizes a consensus DNA sequence, the specific binding energy is used to melt a region of the promoter, part of which serves as a template for the initiation of RNA synthesis (3). Several studies including the crystal structures show that T7 RNA polymerase recognizes a consensus sequence that extends from Ϫ17 to ϩ4 relative to the transcription start site. In the absence of the initiating nucleotide, GTP, T7 RNA polymerase melts Ϫ4 to ϩ2 region, which includes the TATA sequence, and in the presence of GTP, the unpaired region extends from Ϫ4 to ϩ4 (4). Our goal in these studies was to determine the kinetic and thermodynamic parameters that govern the formation of the closed and open complexes. These studies provide the framework to understand how the efficiency of initiation can be regulated by protein-DNA interactions during initiation.Previously, we have used the fluorescent adenine analog 2-AP, 1 which was incorporated in the promoter DNAs, to monitor the dynamics of T7 RNA polymerase interactions with the T7 promoters (5). These studies were carried out with the promoter DNAs in which all five adenines in the Ϫ4 to ϩ4 region were substituted with 2-AP. These previous studies had indicated that T7 RNA polymerase exists in two forms (5). The fast form binds dsDNA at close to a diffusion-limited rate and melts the promoter...

In synthesizing a double-stranded DNA from viral RNA, HIV-1 reverse transcriptase (RT) generates an RNA/DNA intermediate. RT also degrades the RNA strand and synthesizes the second DNA strand. The RNase H active site of RT functions as a nuclease to cleave the RNA strand; however, the structural basis for endonucleolytic cleavage of the RNA strand remains elusive. Here we report crystal structures of RT-RNA/DNA-dATP and RT-RNA/DNA-nevirapine (NVP) ternary complexes at 2.5 and 2.9 Å resolution, respectively. The polymerase region of RT-RNA/DNA-dATP complex resembles DNA/DNA ternary complexes apart from additional interactions of 2′-OH groups of the RNA strand. The conformation and binding of RNA/DNA deviates significantly after the seventh nucleotide versus a DNA/DNA substrate. Binding of NVP slides the RNA/DNA non-uniformly over RT, and the RNA strand moves closer to the RNase H active site. Two additional structures, one containing a gapped RNA and another a bulged RNA, reveal that conformational changes of an RNA/DNA and increased interactions with the RNase H domain, including the interaction of a 2′-OH with N474, help to position the RNA nearer to the active site. The structures and existing biochemical data suggest a nucleic acid conformation-induced mechanism for guiding cleavage of the RNA strand.

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