Smita S. Patel

The helicase from hepatitis C virus is active as an oligomer

The helicase from hepatitis C virus (HCV NS3h) residing on the C-terminal domain of nonstructural protein 3 was considered to be monomeric by several researchers. Here we demonstrate, based on biochemical kinetic data, that the HCV helicase acts as an oligomer. The increase in the ATPase k cat of the NS3h protein with increasing protein concentration provided evidence for oligomerization. A sharp decrease in the unwinding rate was observed when the wild type NS3h was mixed with the ATPase deficient mutants of NS3h protein. This provided strong support for both mixed oligomer formation and subunit interactions for the HCV helicase. Chemical cross-linking of NS3h protein was an inefficient process, but yielded cross-linked protein oligomers of various sizes. The information currently available for HCV helicase is consistent with the hypothesis that oligomers of NS3h are not stable and the helicase subunits exchange during unwinding. Nevertheless, oligomerization of HCV helicase stimulates the ATPase activity, and it is required for the helicase activity.

Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 5'- and 3'-tails of the forked DNA substrate.

Bacteriophage T7 DNA helicase requires two noncomplementary single-stranded DNA (ssDNA) tails next to a double-stranded DNA (dsDNA) region to initiate DNA unwinding. The interactions of the helicase with the DNA were investigated using a series of forked DNAs. Our results show that the helicase interacts asymmetrically with the two tails of the forked DNA. When the helicase was preassembled on the forked DNA before the start of unwinding, a DNA with 15-nucleotide (nt) 3′-tail and 35-nt 5′-tail was unwound with optimal rates close to 60 base pairs/s at 18 °C. When the helicase was not preassembled on the DNA, a >65-nt long 5′-tail was required for maximal unwinding rates of 12 base pairs/s. We show that the helicase interacts specifically with the ssDNA region and maintains contact with both ssDNA strands during DNA unwinding, since conversion of the two ssDNA tails to dsDNA structures greatly inhibited unwinding, and the helicase was unable to unwind past a nick in the dsDNA region. These studies have provided new insights into the mechanism of DNA unwinding. We propose an exclusion model of DNA unwinding in which T7 helicase hexamer interacts mainly with the ssDNA strands during DNA unwinding, encircling the 5′-strand and excluding the 3′-strand from the hole.

Equilibrium and Stopped-flow Kinetic Studies of Interaction between T7 RNA Polymerase and Its Promoters Measured by Protein and 2-Aminopurine Fluorescence Changes

The mechanism of bacteriophage T7 RNA polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods. To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes. The protein fluorescence changes measured conformational changes in the polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (−4 to +4), measured structural changes in the promoter DNA, such as DNA melting. The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism, where the RNA polymerase forms an initial weak EDa complex rapidly with an equilibrium association constant K1. The EDa complex then undergoes a conformational change to EDb, wherein RNA polymerase is specifically and tightly bound to the promoter DNA. Both the polymerase and the promoter DNA may undergo structural changes during this isomerization step. The isomerization of EDa to EDb is a fast step relative to the rate of transcription initiation and its rate does not limit transcription initiation. To understand how T7 RNA polymerase modulates its transcriptional efficiency at various promoters at the level of DNA binding, comparative studies with two natural T7 promoters, Φ10 and Φ3.8, were conducted. The results indicate that kinetics, the bimolecular rate constant of DNA binding, kon (K1k2), and the dissociation rate constant, koff (k−2), and thermodynamics, the equilibrium constants of the two steps (K1 and k2/k−2) both play a role in modulating the transcriptional efficiency at the level of DNA binding. Thus, the 2-fold lower kon, the 4-fold higher koff, and the 2-5-fold weaker equilibrium interactions together make Φ3.8 a weaker promoter relative to Φ10.

Bacteriophage T7 DNA Helicase Binds dTTP, Forms Hexamers, and Binds DNA in the Absence of Mg2+ THE PRESENCE OF dTTP IS SUFFICIENT FOR HEXAMER FORMATION AND DNA BINDING

The role of Mg2+ in dTTP hydrolysis, dTTP binding, hexamer formation, and DNA binding was studied in bacteriophage T7 DNA helicase (4A′ protein). The steady state k cat for the dTTPase activity was 200–300-fold lower in the absence of MgCl2, but theK m was only slightly affected. Direct dTTP binding experiments showed that the K d of dTTP was unaffected, but the stoichiometry of dTTP binding was different in the absence of Mg2+. Two dTTPs were found to bind tightly in the absence of Mg2+ in contrast to three to four in the presence of Mg2+. In the presence of DNA there was little difference in the stoichiometry of dTTP binding to 4A′. These results indicate that Mg2+ is not necessary for dTTP binding, but Mg2+ is required for optimal hydrolysis of dTTP. Gel filtration of 4A′ in the presence of dTTP without Mg2+showed that Mg2+ was not necessary, and dTTP was sufficient for hexamer formation. The hexamers formed in the presence of dTTP without Mg2+ were capable of binding single-stranded DNA. However, the 4A′ hexamers formed in the presence of dTDP with or without Mg2+ did not bind DNA, indicating that hexamer formation itself is not sufficient for DNA binding. The hexamers need to be in the correct conformation, in this case in the dTTP-bound state, to interact with the DNA. Thus, the γ-phosphate of dTTP plays an important role in causing a conformational change in the protein that leads to stable interactions of 4A′ with the DNA.

Kinetic Mechanism of GTP Binding and RNA Synthesis during Transcription Initiation by Bacteriophage T7 RNA Polymerase

We have used stopped-flow and rapid chemical quench-flow methods to investigate the kinetics of the early steps during transcription initiation by bacteriophage T7 RNA polymerase. Most promoters of T7 RNA polymerase initiate with two GTPs. The kinetics of GTP binding was investigated by monitoring the fluorescence changes resulting from GTP binding to polymerase and fluorescent 2-aminopurine-containing promoter DNA complex. SchemeFS1 was determined from studies of T7 Φ10 promoter at 25 °C, where (E·D) n represents the polymerase·DNA complex in different conformations. GTPEand GTPI represent the elongating and initiating GTP molecules incorporated at the +2 and +1 positions, respectively. Our studies show that GTP at the elongation site binds with at least 10-fold tighter affinity than the GTP at the initiation site. Two conformational changes were revealed upon GTP binding to the polymerase·2-aminopurine DNA complex. The first conformational change occurred upon GTP binding to the elongation site. This conformational change was reversible, and studies with partially melted DNA and incorrect NTPs suggested that it may represent a DNA melting and/or base pairing step. A second rate-limiting conformational change whose rate was same as the maximum rate of pppGpG synthesis occurred after two GTPs were bound. As with DNA polymerases, this rate-limiting conformational change probably occurs at each NMP incorporation event and may be involved in proper positioning of the initiation and the elongating GTPs within the polymerase active site to achieve efficient and accurate RNA synthesis.

Transcription Termination Factor Rho Contains Three Noncatalytic Nucleotide Binding Sites

The active form of transcription termination factor rho from Escherichia coli is a homohexamer, but several studies suggest that the six subunits of the hexamer are not functionally identical. Rho has three tight and three weak ATP binding sites. Based on our findings, we propose that the tight nucleotide binding sites are noncatalytic and the weak sites are catalytic. In the presence of RNA, the rho-catalyzed ATPase rate is fast, close to 30 s−1. However, under these conditions the three tightly bound nucleotides dissociate from the rho hexamer at a slow rate of 0.02 s−1, indicating that the three tight nucleotide binding sites of rho do not participate in the fast ATPase turnover. These slowly exchanging nucleotide binding sites of rho are capable of hydrolyzing ATP, but the resulting products (ADP and Pi) bind tightly and dissociate from rho about 1500 times slower than the fast ATPase turnover. Both RNA and excess ATP in solution are necessary for stabilizing nucleotide binding at these sites. In the absence of RNA, or when solution ATP is hydrolyzed to ADP, a faster dissociation of nucleotides was observed. Based on these results, we propose that the rho hexamer is similar to the F1-ATPase and T7 DNA helicase-containing noncatalytic sites that do not participate in the fast ATPase turnover. We propose that the three tight sites on rho are the noncatalytic sites and the three weak sites are the catalytic sites.

The Mechanism of ATP Hydrolysis at the Noncatalytic Sites of the Transcription Termination Factor Rho

Escherichia coli transcription termination factor rho is a hexamer with three catalytic subunits that turnover ATP at a fast rate and three noncatalytic subunits that turnover ATP at a relatively slow rate. The mechanism of the ATPase reaction at the noncatalytic sites was determined and was compared with the ATPase mechanism at the catalytic sites. A sequential mechanism for ATP binding or hydrolysis that was proposed for the catalytic sites was not observed at the noncatalytic sites. Pre-steady-state pulse-chase experiments showed that three ATPs were tightly bound to the noncatalytic sites and these were simultaneously hydrolyzed at a rate of 1.8 s−1 at 18 °C. The apparent bimolecular rate constant for ATP binding was determined as 5.4 × 105 m −1 s−1 in the presence of poly(C) RNA. The ATP hydrolysis products dissociated from the noncatalytic sites at 0.02 s−1. The hydrolysis of ATP at the noncatalytic sites was at least 130 times slower, and the overall ATPase turnover was 1500 times slower than that at the catalytic sites. These results from studies of the rho protein are likely to be general to hexameric helicases. We propose that the ATPase activity at the noncatalytic site is too slow to drive translocation of the protein on the nucleic acid or to provide energy for nucleic acid unwinding.