Reverse Transcriptase in Action: FRET-Based Assay for Monitoring Flipping and Polymerase Activity in Real Time.

Reverse transcriptase (RT) of human immunodeficiency virus-1 (HIV-1) is a multifunctional enzyme that catalyzes the conversion of the single stranded viral RNA genome into double-stranded DNA, competent for host-cell integration. RT is endowed with RNA- and DNA-dependent DNA polymerase activity and DNA-directed RNA hydrolysis (RNase H activity). As a key enzyme of reverse transcription, RT is a key target of currently used highly active antiretroviral therapy (HAART), though RT inhibitors offer generally a poor resistance profile, urging new RT inhibitors to be developed. Using single molecule fluorescence approaches, it has been recently shown that RT binding orientation and dynamics on its substrate play a critical role in its activity. Currently, most in vitro RT activity assays, inherently end-point measurements, are based on the detection of reaction products by using radio-labeled or chemically modified nucleotides. Here, we propose a simple and continuous real-time Förster resonance energy transfer (FRET) based-assay for the direct measurement of RT's binding orientation and polymerase activity, with the use of conventional steady-state fluorescence spectroscopy. Under our working conditions, the change in binding orientation and the primer elongation step can be visualized separately on the basis of their opposite fluorescence changes and their different kinetics. The assay presented can easily discriminate non-nucleoside RT inhibitors from nucleoside RT inhibitors and determine reliably their potency. This one-step and one-pot assay constitutes an improved alternative to the currently used screening assays to disclose new anti-RT drugs and identify at the same time the class to which they belong.

Refined model for primer/template binding by HIV-1 reverse transcriptase: pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation events distinguish between different binding modes depending on the nature of the nucleic acid substrate.

The kinetic mechanism of nucleic acid substrate binding and nucleotide incorporation by human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was analysed using synthetic DNA/DNA and DNA/RNA primer/templates (p/t) without predicted secondary structures in the single-stranded region. Determination of the pre-steady-state kinetics of p/t binding by a combination of stopped-flow and quench flow methods indicate a branched binding mechanism for the HIV-1 RT/nucleic acid interaction. Analysis of p/t-RT association by stopped-flow measurements suggest a three-step binding mode with an initial second-order step followed by two isomerisation steps with rates of about 6 s(-1)and 0.5 s(-1), respectively. Determination of the rate-limiting step of the association process via single turnover, single nucleotide incorporation analysis by quench flow measurements revealed two binding events (the initial second-order step cannot be detected with this experimental set-up) with rates of 4 - 7 s(-1)and 0.4 - 0. 7 s(-1), respectively, indicating that both binding events exist in parallel. Thorough pre-steady-state analysis of single turnover, single nucleotide incorporation kinetics showed that dNTP incorporation occurs with a biphasic exponential burst followed by a linear phase. The exponential burst consists of a fast phase with rates of 20 - 60 s(-1) and a slow phase with rates of 0.5 - 2 s(-1), respectively. The relative distribution of these two burst amplitudes differs significantly depending upon which substrate is used. The DNA/RNA-RT complex shows primarily fast incorporation (>80 %) whereas less than 45 % of the DNA/DNA-RT complex incorporate dNTP rapidly. The same relative distribution of amplitudes concerning the two substrates is also found for the association process of RT and p/t. Analysis of dNTP incorporation of the preformed RT-p/t complex in the presence of a nucleic acid competitor shows no effect on the biphasic burst amplitude, however the linear phase disappears. Here, a refined model of the mechanism of RT-p/t binding is presented which is based on the suggestion that two different RT-p/t complexes are formed, i.e. a productive enzyme/substrate complex which is capable of nucleotide incorporation and a non-productive complex which has to undergo an isomerisation before dNTP incorporation can occur. In addition, binding of RT to its substrate can lead to a dead end complex that is not capable of dNTP incorporation.

Temperature-dependent equilibrium between the open and closed conformation of the p66 subunit of HIV-1 reverse transcriptase revealed by site-directed spin labelling.

X-ray crystallographic studies of human immunodeficiency virus type 1 reverse transcriptase complexed with or without substrates or inhibitors show that the heterodimeric enzyme adopts distinct conformations that differ in the orientation of the so-called thumb subdomain in the large subunit. Site-directed spin labelling of mutated residue positions W24C and K287C is applied here to determine the distances between the fingers and thumb subdomains of liganded and unliganded RT in solution. The inter-spin distances of a DNA/DNA and a pseudoknot RNA complexed reverse transcriptase in solution was found to agree with the respective crystal data of the open and closed conformations. For the unliganded reverse transcriptase a temperature-dependent equilibrium between these two states was observed. The fraction of the closed conformation decreased from 95% at 313 K to 65% at 273 K. The spectral separation between the two structures was facilitated by the use of a perdeuterated ([15)N]nitroxide methane-thiosulfonate spin label.

Characterization of the dimerization process of HIV-1 reverse transcriptase heterodimer using intrinsic protein fluorescence.

Intrinsic protein fluorescence has been used to study dimerization of the HIV-1 reverse transcriptase (RT). We observed a 25% increase of the tryptophan fluorescence of the enzyme during dissociation of the subunits induced by the addition of acetonitrile. Upon reassociation of the separated subunits, the original fluorescence emission of the heterodimer is restored. A two-state transition model for the RT dimerization process in which the dimers are in equilibrium with folded monomers is proposed. The free energy of dissociation was determined to be 12.2 (+/- 0.2) kcal/mol. In the absence of Mg2+ ions a decrease of this value was observed, whereas the addition of a synthetic primer/template (18/36mer) results in an increase of dimer stability. Analyzing the effect of Mg2+ on the establishment of the binding equilibrium, a dramatic effect with a 100-fold acceleration of the association by the divalent ion was observed.

Factors contributing to the inhibition of HIV reverse transcriptase by chain-terminating nucleotides in vitro and in vivo.

Arguments are presented leading to the conclusion that two major factors contribute to the potency of inhibition of DNA-polymerase activity by chain-terminating nucleotides. The relative significance of these factors varies with the reaction conditions, particularly with the length of the template and the concentration ratio of enzyme (reverse transcriptase or other DNA polymerase) to primer. It is concluded that potent inhibition of HIV-reverse transcriptase activity under typical in vitro and in vivo conditions arises from different features of the interaction of chain terminators with the enzyme. A new method of testing for the parameter important under in vivo conditions is suggested.

RNase H activity of HIV reverse transcriptases is confined exclusively to the dimeric forms.

A method for the rapid preparation of a defined substrate to monitor RNase H activity has been developed. Using this substrate, we have investigated the RNase H activities of the different forms of recombinant HIV-1 and HIV-2 reverse transcriptase (RT) in detail. As we report here, RNase H activity is associated only with the dimeric forms (p51/p66 or p66/p66) of the enzymes.

Peptide-mediated cellular delivery of oligonucleotide-based therapeutics in vitro: quantitative evaluation of overall efficacy employing easy to handle reporter systems.

Cellular uptake of therapeutic oligonucleotides and subsequent intracellular trafficking to their target sites represents the major technical hurdle for the biological effectiveness of these potential drugs. Accordingly, laboratories worldwide focus on the development of suitable delivery systems. Among the different available non-viral systems like cationic polymers, cationic liposomes and polymeric nanoparticles, cell-penetrating peptides (CPPs) represent an attractive concept to bypass the problem of poor membrane permeability of these charged macromolecules. While uptake per se in most cases does not represent the main obstacle of nucleic acid delivery in vitro, it becomes increasingly apparent that intracellular trafficking is the bottleneck. As a consequence, in order to optimize a given delivery system, a side-by-side analysis of nucleic acid cargo internalized and the corresponding biological effect is required to determine the overall efficacy. In this review, we will concentrate on peptide-mediated delivery of siRNAs and steric block oligonucleotides and discuss different methods for quantitative assessment of the amount of cargo taken up and how to correlate those numbers with biological effects by applying easy to handle reporter systems. To illustrate current limitations of non-viral nucleic acid delivery systems, we present own data as an example and discuss options of how to enhance trafficking of molecules entrapped in cellular compartments.

Dimerization of human immunodeficiency virus type 1 reverse transcriptase. A target for chemotherapeutic intervention.

Recombinant human immunodeficiency virus type 1 reverse transcriptase has been used to investigate the process of dimer formation and the properties of the different mono- and dimeric forms of the enzyme. The studies show that reverse transcriptase activity is exclusively confined to the dimeric forms. As we also demonstrate, the association rate constant between the monomers is relatively low so that the dimer-monomer equilibrium is very slowly established. This offers a new and potentially interesting target for antiviral chemotherapy with presumably higher specificity than the currently used nucleoside analogs (Yarchoan, R., Mitsuya, H., Myers, C.E., and Broder, S. (1989) N. Eng. J. Med. 321, 726-738), which in their active triphosphorylated form are also inhibitors of cellular polymerases.

Interaction of fluorescently labeled dideoxynucleotides with HIV-1 reverse transcriptase.

Succinylfluorescein-labeled dideoxyTTP has been used as a substrate for reverse transcriptase from HIV-1. On addition to the 3'-end of a primer molecule, there is a reduction of fluorescence yield of a factor of ca. 4. Release of a fluorescent DNA/DNA primer/template duplex from its complex with reverse transcriptase results in a reduction of fluorescence by a further factor of 2. The fluorescent nucleotide is incorporated somewhat less efficiently than 3'-azidoTMP and TMP, which show similar incorporation kinetics. Fluorescent chain-terminated primers have been used to investigate the interaction of normal and chain-terminated primer/template complexes with reverse transcriptase. The dissociation constant of a 36/18-mer was 0.65 nM, whereas that of the same complex after the addition of the fluorescent chain-terminating nucleotide to the primer was 3 nM at 25 degrees C. The rate of dissociation of the latter complex from the enzyme was 0.04 s-1. This was decreased by a factor of ca. 10 at high concentrations (greater than 200 microM) of the nucleotide triphosphate complementary to the next position of the template. The results obtained suggest that potent inhibition of reverse transcriptase activity in in vitro assays results from formation of a slowly dissociating complex between the enzyme and chain-terminated primer/template complexes. However, arguments are presented that lead to the conclusion that this is not the mode of inhibition in cells invaded by HIV. At the prevailing relative concentrations in this situation, chain termination resulting in incomplete transcription is likely to be the major factor.

RNA helicase activity associated with the human p68 protein.

It has been proposed that p68, a nuclear protein of relative molecular mass 68,000, functions in the regulation of cell growth and division. A complementary DNA analysis of the protein has revealed extensive amino-acid sequence homology to the products of a set of genes recently identified in organisms as diverse as Escherichia coli and man, which include the eukaryotic translation initiation factor elF-4A. The protein products of the new gene family have several motifs in common which are thought to be involved in nucleic acid unwinding. As yet, however, only elF-4A, through its effect on RNA, has been shown to possess unwinding activity. Here we report that purified p68 also exhibits RNA-dependent ATPase activity and functions as an RNA helicase in vitro. The protein was first identified by its specific immunological cross reaction with the simian virus 40 large T antigen, the transforming protein of a small DNA tumour virus. Surprisingly, T antigen also has an RNA-unwinding activity: the homology between the two polypeptides, although confined to only a small region resembling the epitope of the cross-reacting antibody (PAb204), should therefore be of functional significance. Furthermore, the RNA-unwinding activity may be involved in the growth-regulating functions of both proteins.

Expression of the heterodimeric form of human immunodeficiency virus type 2 reverse transcriptase in Escherichia coli and characterization of the enzyme.

A system for the expression of recombinant human immunodeficiency virus type 2 (HIV-2) reverse transcriptase (RT) in Escherichia coli has been developed, which allows purification of the heterodimeric form of the enzyme as well as the separate purification of the two subunits. It is shown that equilibrium formation between monomeric and homodimeric forms of the recombinant 66- and 51-kDa subunits is considerably more rapid than in the case of the corresponding homodimeric forms of HIV-1 RT. In accordance with our previously published studies on HIV-1 RT (Restle, T., Müller, B., and Goody, R.S. (1990) J. Biol. Chem. 265, 8986-8988) RNA-dependent DNA polymerase activity of the HIV-2 RT preparations can be exactly correlated to their dimer content. No significant heterodimer formation can be observed upon coexpression of the 66-kDa subunit of HIV-2 RT with the 51-kDa subunit of HIV-1 RT in the same cell, indicating differences in the dimerization domains of the two proteins. Recombinant HIV-2 RT is not recognized by a set of 23 monoclonal antibodies raised against HIV-1 RT, although it shows weak cross-reactivity with sera from HIV-1-infected patients.

The structure of HIV-1 reverse transcriptase complexed with an RNA pseudoknot inhibitor.

Small RNA pseudoknots, selected to bind human immunodeficiency virus type 1 (HIV-1) reverse transcriptase tightly, are potent inhibitors of reverse transcriptase. The co-crystal structure of reverse transcriptase complexed with a 33 nucleotide RNA pseudoknot has been determined by fitting the ligand into a high quality, 4-fold averaged 4.8 A resolution electron density map. The RNA is kinked between stems S1 and S2, thereby optimizing its contacts with subunits of the heterodimer. Its binding site extends along the cleft that lies between the polymerase and RNase H active sites, partially overlaps with that observed for duplex DNA and presumably overlaps some portion of the tRNA site. Stem S2 and loop L1 stabilize the 'closed' conformation of the polymerase through extensive electrostatic interactions with several basic residues in helix I of the p66 thumb and in the p66 fingers domain. Presumably, this RNA ligand inhibits reverse transcriptase by binding to a site that partly overlaps the primer-template binding site.

Inhibition of human immunodeficiency virus type 1 reverse transcriptase dimerization using synthetic peptides derived from the connection domain.

Based on presently available information on the structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, peptides have been synthesized which correspond to the sequence of a particular region of the protein involved in formation of the active heterodimeric form of the enzyme. Several peptides that are 15-19 amino acids long and that are derived from the so-called connection domain of the reverse transcriptase are able to inhibit dimerization of the enzyme and thus inhibit development of its enzymatic activities. In particular, a tryptophan-rich 19-mer corresponding to residues 389-407 was relatively efficient, showing an apparent dissociation constant in the micromolar range for one or both of the subunits. The sequence of this region is identical for both subunits, since one (molecular mass of 51 kDa) is the proteolytic product of the other (molecular mass of 66 kDa). Dissociation of the preformed heterodimer could not be induced by the peptides, but increasing concentrations reduced the rate of dimerization in a concentration-dependent manner until it became immeasurable at high concentrations. The results suggest that inhibition of dimerization of reverse transcriptase is an attractive approach to chemotherapeutic intervention in HIV infection and that further development of peptide-based inhibition strategies is worth pursuing.

Co-expression of the subunits of the heterodimer of HIV-1 reverse transcriptase in Escherichia coli.

Expression of the 66-kDa form of human immunodeficiency virus, type 1 reverse transcriptase in Escherichia coli leads to isolation of small amounts of a 2 x 66-kDa homodimer and larger amounts of a heterodimer form of the enzyme in which the 66-kDa protein is complexed with its carboxyl-terminally truncated is complexed with its carboxyl-terminally truncated 51-kDa form. The latter arises via proteolysis by contaminating proteases. The heterodimer, which was characterized by gel filtration (apparent native molecular mass of 120-130 kDa), was the most active form of the enzyme (specific activity, 5000 units/mg, cf. less than 2000 for the 66-kDa fragment). The 66-kDa fragment alone was shown to be only partially dimerized, with the activity residing mainly in the dimer fraction. Proteolysis of the 66-kDa form resulting partially in the 51-kDa form led to an increase in reverse transcriptase activity. Expression of a truncated version of the protein containing the first 428 amino acids of the reverse transcriptase coding region led to a protein which had low but measurable reverse transcriptase activity (400-500 units/mg). Co-expression of the two proteins on a single plasmid led to expression in a 1:1 ratio. The 1:1 mixture behaved as a heterodimer, as shown by its chromatographic properties. It is likely that the mechanism for the production of heterodimers in vivo involves cleavage of 66-kDa monomers followed by rapid dimerization of the 51- and 66-kDa forms to give the heterodimeric form, which is stable toward further proteolysis.

Structure-function relationships of HIV-1 reverse transcriptase determined using monoclonal antibodies.

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is one of the main targets in approaches to the chemotherapy of AIDS. A detailed knowledge of structure-function relationships of this enzyme is a prerequisite for rational drug design. We have used monoclonal antibodies as tools to identify functionally important regions of the protein. The preparation of 23 murine monoclonal antibodies (mAb) against HIV-1 reverse transcriptase and their different effects on the enzyme are described. The interaction of purified mAbs with HIV-1 RT was demonstrated by enzyme-linked immunosorbent assay (ELISA), Western blots, and high performance liquid chromatography size exclusion chromatography. One of the antibodies also recognized recombinant HIV-2 RT. Antibody binding epitopes on HIV-1 RT were analyzed by immunoblotting using cyanogen bromide fragmented RT, C-terminally truncated mutants, and a peptide ELISA employing 15-mer synthetic overlapping peptides spanning nearly the complete polypeptide chain. The epitopes were mapped within three domains corresponding to amino acids 200-230, 300-428, and 528-560. Two mAbs show neutralizing properties on enzymatic functions of RT. One affects the polymerase activity and to a certain degree the RNase H activity of the enzyme, whereas the other inhibits the latter activity exclusively. mAb 28, which blocks the polymerase activity, interferes with the nucleotide binding region of RT, as shown by fluorescence spectroscopy using a labeled template/primer complex. By investigating the antibody effects on dimer formation of the heterodimeric enzyme, three domains corresponding to amino acids 230-300, 350-428, and residues around amino acid 540 involved in protein-protein interactions were localized.

Conformational stability of dimeric HIV-1 and HIV-2 reverse transcriptases.

The dissociation of dimeric reverse transcriptase (RT) of the human immunodeficiency virus (HIV) types 1 and 2 has been investigated using acetonitrile as a dissociating agent. The equilibrium transitions were monitored by combining different approaches (fluorescence spectroscopy, polymerase activity assay, and size-exclusion HPLC). The dissociation of RT induced a complete loss of polymerase activity and a 25% increase of the intrinsic fluorescence. It is fully reversible, and the midpoints of the equilibrium transition curves are dependent on the concentration of the enzyme used, suggesting a two-state transition model for the dissociation of RT in which dimers are in equilibrium with folded monomers. For both RTs, the heterodimeric form is more stable against dissociating agents and different pH than the corresponding homodimeric form. Moreover, heterodimeric HIV-2 RT exhibits a higher stability than HIV-1 RT, with a free energy of dissociation of 12.1 kcal/mol at pH 6.5 and 25 degrees C, instead of 10 kcal/mol for HIV-1 RT. The binding of a primer/template induces a marked conformational change in both RTs, shown by the lower accessibility of the tryptophans to quenchers and the increase in tryptophan heterogeneity, and stabilized the dimeric form of both RTs (10-100-fold). The central role of hydrophobic interactions in dimer formation has been revealed by the 30% increase of exposure of the tryptophan cluster to quenchers upon dissociation of RT and the binding of 4 equiv of 1-anilino-8-naphthalenesulfonate to the dissociated enzymes.

HIV-1 reverse transcriptase-pseudoknot RNA aptamer interaction has a binding affinity in the low picomolar range coupled with high specificity.

Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the identification of small oligonucleotides that bind with high affinity and specificity to target proteins. Such DNAs/RNAs are a new class of potential chemotherapeutics that could block the enzymatic activity of pathologically relevant proteins. We have conducted a detailed biochemical study of the interaction of human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) with a SELEX-derived pseudoknot RNA aptamer. Electron paramagnetic resonance spectroscopy of site-directed spin-labeled RT mutants revealed that this aptamer was selected for binding to the "closed" conformation of the enzyme. Kinetic analysis showed that the RNA inhibitor bound to HIV RT in a two-step process, with association rates similar to those described for model DNA/DNA and DNA/RNA substrates. However, the dissociation of the pseudoknot RNA from RT was dramatically slower than observed for model substrates. Equilibrium binding studies revealed an extraordinarily low K(d), of about 25 pm, for the enzyme-aptamer interaction, presumably a consequence of the slow off-rates. Additionally, this pseudoknot aptamer is highly specific for HIV-1 RT, with the closely related HIV-2 enzyme showing a binding affinity close to 4 orders of magnitude lower.

Kinetics of interaction of HIV reverse transcriptase with primer/template.

Intrinsic protein fluorescence of reverse transcriptases from HIV-1 and HIV-2 provides a sensitive signal for monitoring the interaction of the enzymes with primer/template duplex molecules. Kd values for 18/36-mer DNA/DNA duplexes were found to be in the range of a few nanomolar (about 3 times higher for the enzyme from HIV-2 than for that from HIV-1). The quenching of protein fluorescence induced on binding primer/template, together with an increase in extrinsic fluorescence on interaction with primer/template containing a fluorescent nucleotide at the 3'-end of the primer, was used to investigate the kinetics of interaction with reverse transcriptase from HIV-1. The results can be explained in terms of a two-step binding model, with a rapid diffusion-limited initial association (k(ass) = ca. 5 x 10(8) M-1 s-1) followed by a slow isomerization step (k = ca. 0.5 s-1). These (forward) rate constants are increased in the presence of a non-nucleoside inhibitor (S-TIBO) of HIV-1 reverse transcriptase, while the reverse rate constant for the second step is decreased, leading to an increase in affinity between the enzyme and primer/template by a factor of at least 10 when S-TIBO is bound. The results are discussed in terms of present knowledge of the structure of reverse transcriptase.

Analysis of the polymerization kinetics of homodimeric EIAV p51/51 reverse transcriptase implies the formation of a polymerase active site identical to heterodimeric EIAV p66/51 reverse transcriptase.

Homodimeric EIAV p51/51 and heterodimeric EIAV p66/51 reverse transcriptase were purified in order to compare the different modes of DNA synthesis supported by the enzymes. Analysis of the dimerization behavior of the EIAV enzymes indicates that the dimer stability of EIAV reverse transcriptase enzymes is higher than that of their HIV-1 reverse transcriptase counterparts. EIAV p51/51 polymerizes DNA distributively whereas DNA synthesis by EIAV p66/51 is processive. Steady-state and pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation were performed with both enzymes to determine the reasons for the different polymerization behavior. Equilibrium fluorescence titrations demonstrated that the Kd values of EIAV p51/51 for binding of DNA/DNA and DNA/RNA substrates are increased 10-fold and 28-fold, respectively, as compared to EIAV p66/51. Stopped-flow measurements with DNA/DNA show that the increase in the Kd is in part due to a 17. 4-fold higher dissociation rate constant (k-1) for EIAV p51/51. Additionally, with EIAV p51/51, kdiss is increased 7-fold for DNA/DNA and 14-fold for DNA/RNA primer/template substrates, respectively. The lack of the RNase H domain in EIAV p51/51 leads to differences in the pre-steady-state kinetics of nucleotide incorporation on DNA/DNA and DNA/RNA templates. The burst of both enzymes is composed of two phases for both substrates, and the values for the corresponding pre-steady-state burst rates, kpol1 and kpol2, are similar for both enzymes, implying the formation of identical polymerase active sites. However, the amplitudes of the two phases differ with DNA/DNA templates, indicating a different distribution between two states varying greatly in their kinetic competence.

Pre-steady-state kinetic characterization of RNA-primed initiation of transcription by HIV-1 reverse transcriptase and analysis of the transition to a processive DNA-primed polymerization mode.

Single-turnover and equilibrium measurements were carried out to determine the basis of the apparently slow, nonprocessive polymerization reaction catalyzed by HIV-1 reverse transcriptase (RT) during transcription initiation, when both the primer and template are composed of RNA. Comparison of the binding and kinetic parameters of a 20-mer, all-RNA primer/35-mer template substrate to one identical in sequence but composed of a 20-mer, all-DNA primer/35-mer RNA template reveals striking differences. Equilibrium titrations yielded a dissociation constant (Kd) >200 nM for the RNA/RNA-RT complex which is at least 200-fold higher than that of the DNA/RNA-substrate (Kd approximately 1 nM). The affinity of the RT-RNA/RNA complex for dTTP was found to be at least 500 times lower (Kd approximately 3.4 mM) than that of the RT-DNA/RNA complex (Kd approximately 6.6 microM). The single-turnover dNTP incorporation time course using the RNA-primer substrate, the DNA-primer substrate, or a series of RNA-primer substrates preextended with one to eight deoxynucleotides showed that dNTP incorporation occurs with a biphasic exponential burst of +1 extension product, followed by a linear phase. At least three different RT-bound forms of the p/ts exist: a fast, kinetically competent form (single-turnover rate approximately 10-50 s-1); a slow form (rate approximately 0.3-1 s-1); and a form that is dead-end (no turnover). The studies further revealed that a switch to a fast, kinetically competent p/t occurs after six dNTPs are incorporated into the RNA primer, with the switch being defined as the transition from a minority to a majority of the p/t bound in the optimal manner.