Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding.

Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these 'macromolecular machines'. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3'-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2-3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3'-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and 'DNA map') for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex.

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity.

We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5'-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex.

Breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork.

We previously used changes in the near-UV circular dichroism and fluorescence spectra of DNA base analogue probes placed site specifically to show that the first three base pairs at the fork junction in model replication fork constructs are significantly opened by "breathing" fluctuations under physiological conditions. Here, we use these probes to provide mechanistic snapshots of the initial interactions of the DNA fork with a tight-binding replication helicase in solution. The primosome helicase of bacteriophage T4 was assembled from six (gp41) helicase subunits, one (gp61) primase subunit, and nonhydrolyzable GTPγS. When bound to a DNA replication fork construct this complex advances one base pair into the duplex portion of the fork and forms a stably bound helicase "initiation complex." Replacement of GTPγS with GTP permits the completion of the helicase-driven unwinding process. Our spectroscopic probes show that the primosome in this stable helicase initiation complex binds the DNA of the fork primarily via backbone contacts and holds the first complementary base pair of the fork in an open conformation, whereas the second, third, and fourth base pairs of the duplex show essentially the breathing behavior that previously characterized the first three base pairs of the free fork. These spectral changes, together with dynamic fluorescence quenching results, are consistent with a primosome-binding model in which the lagging DNA strand passes through the central hole of the hexagonal helicase, the leading strand binds to the "outside" surfaces of subunits of the helicase hexamer, and the single primase subunit interacts with both strands.

Assembly and subunit stoichiometry of the functional helicase-primase (primosome) complex of bacteriophage T4.

Physical biochemical techniques are used to establish the structure, subunit stoichiometry, and assembly pathway of the primosome complex of the bacteriophage T4 DNA replication system. Analytical ultracentrifugation and fluorescence anisotropy methods show that the functional T4 primosome consists of six gp41 helicase subunits that assemble into a hexagon, driven by the binding of six NTPs (or six nonhydrolyzable GTPγS analogues) that are located at and stabilize the intersubunit interfaces, together with a single tightly bound gp61 primase subunit. Assembling the components of the primosome onto a model DNA replication fork is a multistep process, but equilibrium cannot be reached along all mixing pathways. Producing a functional complex requires that the helicase hexamer be assembled in the presence of the DNA replication fork construct prior to the addition of the primase to avoid the formation of metastable DNA-protein aggregates. The gp41 helicase hexamer binds weakly to fork DNA in the absence of primase, but forms a much more stable primosome complex that expresses full and functional helicase (and primase) activities when bound to a gp61 primase subunit at a helicase:primase subunit ratio of 61. The presence of additional primase subunits does not change the molecular mass or helicase activity of the primosome, but significantly inhibits its primase activity. We develop both an assembly pathway and a minimal mechanistic model for the structure and function of the T4 primosome that are likely to be relevant to the assembly and function of the replication primosome subassemblies of higher organisms as well.

Development of a "modular" scheme to describe the kinetics of transcript elongation by RNA polymerase.

Transcript elongation by RNA polymerase involves the sequential appearance of several alternative and off-pathway states of the transcript elongation complex (TEC), and this complicates modeling of the kinetics of the transcription elongation process. Based on solutions of the chemical master equation for such transcription systems as a function of time, we here develop a modular scheme for simulating such kinetic transcription data. This scheme deals explicitly with the problem of TEC desynchronization as transcript synthesis proceeds, and develops kinetic modules to permit the various alternative states of the TECs (paused states, backtracked states, arrested states, and terminated states) to be introduced one-by-one as needed. In this way, we can set up a comprehensive kinetic model of appropriate complexity to fit the known transcriptional properties of any given DNA template and set of experimental conditions, including regulatory cofactors. In the companion article, this modular scheme is successfully used to model kinetic transcription elongation data obtained by bulk-gel electrophoresis quenching procedures and real-time surface plasmon resonance methods from a template of known sequence that contains defined pause, stall, and termination sites.

Fitting experimental transcription data with a comprehensive template-dependent modular kinetic model.

In the companion article, we developed a modular scheme for representing the kinetics of transcription elongation by RNA polymerase. As an example of how to use these approaches, in this article we use a comprehensive modular model of this sort to fit experimental transcript elongation results obtained on the canonical tR2 template of phage λ by means of complementary bulk gel electrophoresis and surface plasmon resonance assays. The gel electrophoresis results, obtained in experiments quenched at various times after initiation of transcription, provide distributions of RNA lengths as a function of time. The surface plasmon resonance methods were used to monitor increases and decreases in the total mass of transcription elongation complexes in the same experiments. The different measures of transcription dynamics that these methods provide allow us to use them in combination to obtain a set of largely robust and well-defined kinetic parameters. The results show that our modular approach can be used to develop and test predictive kinetic schemes that can be fit to real transcription elongation data. They also suggest that these approaches can be extended to simulate the kinetics of other processes that involve the processive extension or shortening of nucleic acid chains and related systems of sequential branching reaction events.

Monitoring RNA transcription in real time by using surface plasmon resonance.

The decision to elongate or terminate the RNA chain at specific DNA template positions during transcription is kinetically regulated, but the methods used to measure the rates of these processes have not been sufficiently quantitative to permit detailed mechanistic analysis of the steps involved. Here, we use surface plasmon resonance (SPR) technology to monitor RNA transcription by Escherichia coli RNA polymerase (RNAP) in solution and in real time. We show that binding of RNAP to immobilized DNA templates to form active initiation or elongation complexes can be resolved and monitored by this method, and that changes during transcription that involve the gain or loss of bound mass, including the release of the sigma factor during the initiation-elongation transition, the synthesis of the RNA transcript, and the release of core RNAP and nascent RNA at intrinsic terminators, can all be observed. The SPR method also permits the discrimination of released termination products from paused and other intermediate complexes at terminators. We have used this approach to show that the rate constant for transcript release at intrinsic terminators tR2 and tR' is approximately 2-3 s(-1) and that the extent of release at these terminators is consistent with known termination efficiencies. Simulation techniques have been used to fit the measured parameters to a simple kinetic model of transcription and the implications of these results for transcriptional regulation are discussed.

The antitermination activity of bacteriophage lambda N protein is controlled by the kinetics of an RNA-looping-facilitated interaction with the transcription complex.

Protein N of bacteriophage lambda activates the lytic phase of phage development in infected Escherichia coli cells by suppressing the activity of transcriptional terminators that prevent the synthesis of essential phage proteins. N binds tightly to the boxB RNA hairpin located near the 5' end of the nascent pL and pR transcripts and induces an antitermination response in the RNA polymerase (RNAP) of elongation complexes located at terminators far downstream. Here we test an RNA looping model for this N-dependent "action at a distance" by cleaving the nascent transcript between boxB and RNAP during transcript elongation. Cleavage decreases antitermination, showing that an intact RNA transcript is required to stabilize the interaction of boxB-bound N with RNAP during transcription. In contrast, an antitermination complex that also contains Nus factors retains N-dependent activity after transcript cleavage, suggesting that these host factors further stabilize the N-RNAP interaction. Thus, the binding of N alone to RNAP is controlled by an RNA looping equilibrium, but after formation of the initial RNA loop and in the presence of Nus factors the system no longer equilibrates on the transcription time scale, meaning that the "range" of antitermination activity along the template in the full antitermination system is kinetically controlled by the dissociation rate of the stabilized N-RNAP complex. Theoretical calculations of nucleic acid end-to-end contact probabilities are used to estimate the local concentrations of boxB-bound N at elongation complexes poised at terminators, and are combined with N activity measurements at various boxB-to-terminator distances to obtain an intrinsic affinity (K(d)) of approximately 2 x 10(-5) M for the N-RNAP interaction. This RNA looping approach is extended to include the effects of N binding at nonspecific RNA sites on the transcript and the implications for transcription control in other regulatory systems are discussed.