Rajiv P. Bandwar

Structural basis for the role of the K65R mutation in HIV-1 reverse transcriptase polymerization, excision antagonism, and tenofovir resistance

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.

Peculiar 2-Aminopurine Fluorescence Monitors the Dynamics of Open Complex Formation by Bacteriophage T7 RNA Polymerase

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 −5, during open complex formation. The unstacking may be a critical event in directing and placing the template strand correctly in the T7 RNA polymerase active site upon promoter melting for template directed RNA synthesis. Based on equilibrium fluorescence and stopped-flow kinetic studies, we propose that a fast form of T7 RNA polymerase binds promoter double-stranded DNA by a three-step mechanism. The initial collision complex or a closed complex, EDc is formed with a K d of 1.8 μm. This complex isomerizes to an open complex, EDo1, in an energetically unfavorable reaction with an equilibrium constant of 0.12. The EDo1 further isomerizes to a more stable open complex, EDo2, with a rate constant around 300 s− 1. Thus, in the absence of the initiating nucleotide, GTP, the overall equilibrium constant for closed to open complex conversion is 0.5 and the net rate of open complex formation is nearly 150 s− 1.

Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase

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 stopped-flow 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.

Extended Upstream A-T Sequence Increases T7 Promoter Strength

Bacteriophage T7 promoters contain a consensus sequence from -17 to +6 relative to the transcription start site, +1. In addition, the strong class III promoters are characterized by an extended AT-rich region upstream of -17, which is often interrupted by one or more GC base pairs in the weaker class II promoters. Herein we studied the role of the AT-rich region upstream of -17 in transcription regulation of T7 RNA polymerase. Equilibrium DNA binding studies with promoter fragments of consensus sequence truncated at various positions between -17 and -27 showed that the polymerase-promoter complex is significantly stabilized as the upstream AT-rich sequence is extended to and beyond -22. Similarly, promoters in which the AT-rich region from -17 to -22 is interrupted by several GC base pairs showed weak binding. Kinetic studies indicated that the presence of extended AT-rich sequence slows the dissociation rate constant of the polymerase-promoter complex and slightly stimulates the association rate constant, thereby increasing the stability of the complex. Measurement of the transcription activity revealed that the extended AT-rich region does not affect the kinetics of abortive synthesis up to the formation of 8-nucleotide RNA but causes accumulation of longer abortive products between 9 and 13 nucleotides. The observed effects of the upstream DNA region were AT sequence-specific, and the results suggested a larger role for the extended AT-rich sequence that has been unappreciated previously. We propose that the AT-rich DNA sequence upstream of -17 plays a role in modulating the efficiency of transcription initiation by affecting both the affinity of T7 RNA polymerase for the promoter and the efficiency of promoter clearance.

Structures of HIV-1 RT-RNA/DNA ternary complexes with dATP and nevirapine reveal conformational flexibility of RNA/DNA: insights into requirements for RNase H cleavage

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.

The energetics of consensus promoter opening by T7 RNA polymerase

The consensus 23 base-pair T7 DNA promoter is classically divided into two domains, an upstream binding domain (-17 to -5), and a downstream initiation domain (-4 to +6) relative to the transcription start site at +1. During transcription initiation, T7 RNA polymerase (T7 RNAP) melts specifically the -4 to +2/+3 (TATAGG/G) region of the duplex DNA promoter to form a pre-initiation open complex. No external energy source is used and the energy for open complex formation is derived from the free energy of specific interactions with the binding domain, particularly the specificity region (-13 to -6). Using 2-aminopurine fluorescence-based equilibrium and kinetic measurements, we have measured the binding affinities of various topologically modified DNA promoters (40 bp in length) that represent initial, final, and transition-state analogs of the promoter DNA in the T7 RNAP-DNA complex, to determine the energy of specific binding interactions, and the energy required for forming an initiation bubble. The results indicate that 16-16.5 kcal mol(-1) of free energy is made available upon T7 RNAP binding (through specificity loop) to the promoter binding domain. To melt the TATAGG/G sequence 7-8 kcal mol(-1) of free energy is utilized; this compares with approximately 6 kcal mol(-1) predicted from nearest neighbor analysis. The remaining 8.5-9.5 kcal mol(-1) of net free energy is retained for stabilization of the specific pre-initiation binary complex. Of the 7-8 kcal mol(-1) energy that is used to generate the pre-initiation DNA bubble in the open complex, we estimate that one half (3.5-4 kcal mol(-1)) is utilized for nucleation/deformation process (through bending, untwisting, etc.) in the melting region (-4 to -1 TATA) of the initiation domain (-4 to +6), and appears to be independent of the nucleation site within this region. The other half is utilized in unpairing the +1 to +2/+3 GG/G sequence for initiation. The interactions of T7 RNAP with a 20-bp non-specific DNA on the other hand are very weak (DeltaG<-5k cal mol(-1)), which is not sufficient to melt and stabilize an open complex of a non-specific DNA.

Kinetics of transcription initiation at lacP1. Multiple roles of cyclic AMP receptor protein.

The cyclic AMP receptor protein (CRP) acts as a transcription activator at many promoters of Escherichia coli. We have examined the kinetics of open complex formation at the lacP1 promoter using tryptophan fluorescence of RNA polymerase and DNA fragments with 2-aminopurine substituted at specific positions. Apart from the closed complex formation and promoter clearance, we were able to detect three steps. The first step after the closed complex formation leads to a rapid increase of 2-aminopurine fluorescence. This was followed by another rapid step in which quenching of tryptophan fluorescence of RNA polymerase was observed. The slowest step detected by 2-aminopurine fluorescence increase is assigned to the final open complex formation. We have found that CRP not only enhances RNA polymerase binding at the promoter, but also enhances the slowest isomerization step by about 2-fold. Furthermore, potassium permanganate probing shows that the conformation of the open complex in the presence of CRP appears qualitatively and quantitatively different from that in the absence of CRP, suggesting that contact with RNA polymerase is maintained throughout the transcription initiation.

THE TRANSITION TO AN ELONGATION COMPLEX BY T7 RNA POLYMERASE IS A MULTISTEP PROCESS

During the transition from an initiation complex to an elongation complex (EC), T7 RNA polymerase undergoes major conformational changes that involve reorientation of a “core” subdomain as a rigid body and extensive refolding of other elements in the 266 residue N-terminal domain. The pathway and timing of these events is poorly understood. To examine this, we introduced proline residues into regions of the N-terminal domain that become α-helical during the reorganization and changed the charge of a key residue that interacts with the RNA:DNA hybrid 5 bp upstream of the active site in the EC but not in the initiation complex. These alterations resulted in a diminished ability to make products >5-7 nt and/or a slow transition through this point. The results indicate that the transition to an EC is a multistep process and that the movement of the core subdomain and reorganization of certain elements in the N-terminal domain commence prior to promoter release (at 8-9 nt).

Fluorescence methods for studying the kinetics and thermodynamics of transcription initiation

Publisher Summary This chapter focuses on transient-state kinetic approaches to elucidate the pathway of transcription initiation. These methods are discussed for T7 RNAP, a 99-kDa single subunit phage polymerase that has been characterized extensively, both structurally and mechanistically. The methods described are general and applicable to the studies of other enzymes and polymerases. Transient-state kinetic experiments are designed to follow the formation and decay of reacting species as a function of time. The concentrations are determined directly by radiometric methods or indirectly through optical changes associated with the formation of intermediates and products. Fluorescence methods are described in this chapter to determine the equilibrium-binding constants, and both fluorescence and radiometric methods to determine the intrinsic rate constants of the transcription initiation pathway. The initial steps of RNAP binding to the promoter DNA and formation of the open complex were measured by the stopped-flow fluorescence method. The steps of nucleotide binding and RNA synthesis were measured by both stopped-flow fluorescence and radiometric rapid chemical-quenched flow methods. The chapter emphasizes on the types of experiments that should be used in conjunction, and the data that should be fit globally by numerical methods.

Relaxed Rotational and Scrunching Changes in P266L Mutant of T7 RNA Polymerase Reduce Short Abortive RNAs while Delaying Transition into Elongation

Abortive cycling is a universal feature of transcription initiation catalyzed by DNA-dependent RNA polymerases (RNAP). In bacteriophage T7 RNAP, mutation of proline 266 to leucine (P266L) in the C-linker region connecting the N-terminal promoter binding domain with the C-terminal catalytic domain drastically reduces short abortive products (4–7 nt) while marginally increasing long abortives (9–11 nt). Here we have investigated the transcription initiation pathway of P266L with the goal of understanding the mechanistic basis for short and long abortive synthesis. We show that the P266L mutation does not alter the affinity for the promoter, mildly affects promoter opening, and increases the +1/+2 GTP K d by 2-fold. However, unlike wild-type T7 RNAP that undergoes stepwise rotation of the promoter binding domain and DNA scrunching during initial transcription, the P266L mutant does not undergo coupled rotational/scrunching movements until 7 nt RNA synthesis. The lack of rotation/scrunching correlates with greater stabilities of the initiation complexes of the P266L and decreased short abortive products. The results indicate that the increased flexibility in the C-linker due to P266L mutation enables T7 RNAP to absorb the stress from the growing RNA:DNA hybrid thereby decreasing short abortive products. Increased C-linker flexibility, however, has an adverse effect of delaying the transition into elongation by 1–2 nt, which gives rise to long abortive products. However, a mutation in the upstream promoter region greatly decreases long abortive products in P266L reactions, rendering the combination of P266L and A-15C promoter a desirable pair for efficient in vitro transcription for RNA production. We conclude that the conformational rigidity in the C-linker region conferred by the proline at position 266 is responsible for the undesirable short abortive products, but the rigidity is critical for efficient promoter clearance and transition into elongation.