Craig T. Martin

Stability of gold nanoparticle-bound DNA toward biological, physical, and chemical agents.

Positively charged trimethylammonium‐modified mixed monolayer protected clusters (MMPCs) interact with DNA by complementary electrostatic binding, serving as efficient DNA delivery systems. The stability of gold nanoparticle‐bound DNA toward biological, physical, and chemical agents is investigated. The MMPC‐bound DNA is efficiently protected from DNAse I digestion and experiences nicking/cleavage‐induced morphology changes with higher concentrations of DNAse I. Significant protection of MMPC‐bound DNA was also observed in a physical sonication assay. However, the MMPC‐bound DNA was found to show enhanced cleavage upon exposure to chemically induced radicals. The latter may indicate that bound DNA is bent and wrapped on the surface of the cationic MMPC.

Binding of the priming nucleotide in the initiation of transcription by T7 RNA polymerase.

Unlike DNA polymerases, an RNA polymerase must initiate transcription de novo, that is binding of the initiating (+1) nucleoside triphosphate must be achieved without benefit of the cooperative binding energetics of an associated primer. Since a single Watson-Crick base pair is not stable in solution, RNA polymerases might be expected to provide additional stabilizing interactions to facilitate binding and positioning of the initiating (priming) nucleoside triphosphate at position +1. Consistent with base-specific stabilizing interactions, of the 17 T7 RNA polymerase promoters in the phage genome, 15 begin with guanine. In this work, we demonstrate that the purine N-7 is important in the utilization of the initial substrate GTP. The fact that on a template encoding AG as the first two bases in the transcript (as in the remaining two of the T7 genome) transcription starts predominantly (but not exclusively) at the G at position +2 additionally implicates the purine O-6 as an important recognition element in the major groove. Finally, results suggest that these interactions serve primarily to positionthe initiating base in the active site. It is proposed that T7 RNA polymerase interacts directly with the Hoogsteen side of the initial priming GTP (most likely via an interaction with an arginine side chain in the protein) to provide the extra stability required at this unique step in transcription.

IDENTIFICATION OF ESSENTIAL AMINO ACIDS WITHIN THE PROPOSED CUA BINDING SITE IN SUBUNIT II OF CYTOCHROME C OXIDASE

To explore the nature of proposed ligands to the CuA center in cytochrome c oxidase, site-directed mutagenesis has been initiated in subunit II of the enzyme. Mutations were introduced into the mitochondrial gene from the yeast Saccharomyces cerevisiae by high velocity microprojectile bombardment. A variety of single amino acid substitutions at each of the proposed cysteine and histidine ligands (His-161, Cys-196, Cys-200, and His-204 in the bovine numbering scheme), as well as at the conserved Met-207, all result in yeast which fails to grow on ethanol/glycerol medium. Similarly, all possible paired exchange Cys,His and Cys,Met mutants show the same phenotype. Furthermore, protein stability is severely reduced as evidenced by both the absence of an absorbance maximum at 600 nm in the spectra of mutant cells and the underaccumulation of subunit II, as observed by immunolabeling of mitochondrial extracts. In the same area of the protein, a variety of amino acid substitutions at one of the carboxylates previously implicated in binding cytochrome c, Glu-198, allow (reduced) growth on ethanol/glycerol medium, with normal intracellular levels of protein. These results suggest that a precise folding environment of the CuA site within subunit II is essential for assembly or stable accumulation of cytochrome c oxidase in yeast.

Observed instability of T7 RNA polymerase elongation complexes can be dominated by collision-induced "bumping".

T7 RNA polymerase elongates RNA at a relatively high rate and can displace many tightly bound protein-DNA complexes. Despite these properties, measurements of the stability of stalled elongation complexes have shown lifetimes that are much shorter than those of the multisubunit RNA polymerases. In this work, we demonstrate that the apparent instability of stalled complexes actually arises from the action of trailing RNA polymerases (traveling in the same direction) displacing the stalled complex. Moreover, the instability caused by collision between two polymerases is position dependent. A second polymerase is blocked from promoter binding when a leading complex is stalled 12 bp or less from the promoter. The trailing complex can bind and make abortive transcripts when the leading complex is between 12 and 20 bp from the promoter, but it cannot displace the first complex since it is in a unstable initiation conformation. Only when the leading complex is stalled more than 20 bp away from the promoter can a second polymerase bind, initiate, and displace the leading complex.

Zinc metalloproteins involved in replication and transcription

RNA polymerase (RPase) from E. coli contains two tightly incorporated Zn(II) ions, while the monomeric RPase from bacteriophage T7 does not contain zinc and does not require Zn(II) in the assay. One of the two Zn(II) ions can be differentially removed from E. coli RPase with p-hydroxymercuriphenylsulfonate (PMPS) combined with EDTA and thiol. The resultant Znl or ZnA RPase shows no alteration in transcription initiation and elongation rate from sigma-specific promoters. Biosynthesis of a Co2 RPase and formation of CoA RPase by similar treatment shows the tetrahedral-type Co(II) d-d absorption bands to be associated only with the Co(II) at the A site with maxima at 760 (epsilon = 800), 710 (epsilon = 900), 602 (epsilon = 1500), and 484 (epsilon = 4000) nm. Sulfur to Co(II) charge transfer bands are present at 350 (epsilon = 9600) and 370 (epsilon = 9500) nm. The absorption characteristics strongly suggest that the A site is a tetrathiolate site. While DNA polymerases do not in general appear to contain zinc, gene 32 protein (g32P) from bacteriophage T4, an accessory protein essential for DNA replication and recombination and translational control in the T4 life cycle, is a Zn(II) metalloprotein and contains 1 gram atom of tightly incorporated Zn(II). PMPS displaces the zinc by reacting with three SH groups. Apo-g32P shows markedly altered DNA binding properties. Co(II) substitution gives a protein with intense d-d transitions typical of a tetrahedral Co(II) complex with absorption maxima at 680 (epsilon = 480), 645 (epsilon = 660), 605 (epsilon = 430), 355 (epsilon = 2250), and 320 (epsilon = 3175) nm. The data support a 3 Cys, 1 His coordination site located in the middle of the DNA binding domain of g32P. Data thus far suggest that the Zn(II) binding sites in multisubunit RNA polymerases and in accessory proteins involved in polynucleotide biosynthesis are more likely to play structural or allosteric (regulatory) roles rather than directly participating in catalysis.

Insights into the mechanism of initial transcription in Escherichia coli RNA polymerase.

Background: Abortive cycling is a key feature of RNA polymerases. Results: Nicks and mismatches have little effect on abortive probabilities. Conclusion: The energetics of the hybrid pushing on the protein, rather than that of bubble expansion, is the primary contributor to abortive cycling. Significance: Abortive cycling arises from the need to couple RNA growth to promoter release in RNA polymerases. It has long been known that during initial transcription of the first 8–10 bases of RNA, complexes are relatively unstable, leading to the release of short abortive RNA transcripts. An early “stressed intermediate” model led to a more specific mechanistic model proposing “scrunching” stress as the basis for the instability. Recent studies in the single subunit T7 RNA polymerase have argued against scrunching as the energetic driving force and instead argue for a model in which pushing of the RNA-DNA hybrid against a protein element associated with promoter binding, while likely driving promoter release, reciprocally leads to instability of the hybrid. In this study, we test these models in the structurally unrelated multisubunit bacterial RNA polymerase. Via the targeted introduction of mismatches and nicks in the DNA, we demonstrate that neither downstream bubble collapse nor compaction/scrunching of either the single-stranded template or nontemplate strands is a major force driving abortive instability (although collapse from the downstream end of the bubble does contribute significantly to the instability of artificially halted complexes). In contrast, pushing of the hybrid against a mobile protein element (σ3.2 in the bacterial enzyme) results in substantially increased abortive instability and is likely the primary energetic contributor to abortive cycling. The results suggest that abortive instability is a by-product of the mechanistic need to couple the energy of nucleotide addition (RNA chain growth) to driving the timed release of promoter contacts during initial transcription.

Evaluation of fluorescence spectroscopy methods for mapping melted regions of DNA along the transcription pathway

Publisher Summary This chapter evaluates the fluorescence Spectroscopy technique for mapping melted regions of DNA along the transcription pathway. Fluorescent base analogs provide a relatively non-perturbing probe of very local structural changes within the DNA and/or RNA. They can be used to map melted bubbles in various static states of transcription, either the initially bound promoter complexes, or through walking experiments, in stalled complexes approximating dynamic movement along the DNA. Lists of fluorescent base analogs that have potential utility in probing regions of melted DNA are tabulated. Beyond the mapping of statically melted regions within various transcription complexes, fluorescence is also ideally suited for stopped-flow kinetic measurements of dynamic changes in those complexes. The chapter illustrates the step by step procedure for sample preparation, and analyzes it through fluorescence spectroscopy. Site-specific incorporation of fluorescent probes requires chemical synthesis, but if substantially longer DNA templates are required, these might be introduced by applications of the polymerase chain reaction, in which the probe is introduced in one of the primers. Thus, this approach should increasingly become useful in analyses of the multi-subunit RNA polymerases.

Dissociation of halted T7 RNA polymerase elongation complexes proceeds via a forward-translocation mechanism

A recent model for the mechanism of intrinsic transcription termination involves dissociation of the RNA from forward-translocated (hypertranslocated) states of the complex [Yarnell WS, Roberts JW (1999) Science, 284:611–615]. The current study demonstrates that halted elongation complexes of T7 RNA polymerase in the absence of termination signals can also dissociate via a forward-translocation mechanism. Shortening of the downstream DNA or the introduction of a stretch of mismatched DNA immediately downstream of the halt site reduces a barrier to forward translocation and correspondingly reduces the lifetime of halted complexes. Conversely, introduction of a cross-link downstream of the halt site increases the same barrier and leads to an increase in complex lifetime. Introduction of a mismatch within the bubble reduces a driving force for forward translocation and correspondingly increases the lifetime of the complex, but only for mismatches at the upstream edge of the bubble, as predicted by the model. Mismatching only the two most upstream of the eight bases in the bubble provides a maximal increase in complex stability, suggesting that dissociation occurs primarily from early forward-translocated states. Finally, addition in trans of an oligonucleotide complementary to the nascent RNA just beyond the hybrid complements the loss of driving force derived from placement of a mismatch within the bubble, confirming the expected additivity of effects. Thus, forward translocation is likely a general mechanism for dissociation of elongation complexes, both in the presence and absence of intrinsic termination signals.

Mechanism of instability in abortive cycling by T7 RNA polymerase

Abortive transcription, the premature release of short transcripts 2–8 bases in length, is a unique feature of transcription, accompanying the transition from initiation to elongation in all RNA polymerases. The current study focuses on major factors that relate to the stability of initially transcribing abortive complexes in T7 RNA polymerase. Building on previous studies, results reveal that collapse of the DNA from the downstream end of the bubble is a major contributor to the characteristic instability of abortive complexes. Furthermore, transcription from a novel DNA construct containing a nick between positions –14 and –13 of the nontemplate strand suggests that the more flexible promoter reduces somewhat the strain inherent in initially transcribing complexes, with a resulting decrease in abortive product release. Finally, as assessed by exonuclease III footprinting and transcription profiles, a DNA construct defective in bubble collapse specifically from the downstream end exhibits less abortive cycling and little perturbation of the final transition to elongation, including the process of promoter release.

New Insights into the Mechanism of Initial Transcription THE T7 RNA POLYMERASE MUTANT P266L TRANSITIONS TO ELONGATION AT LONGER RNA LENGTHS THAN WILD TYPE

Background: RNA polymerases must couple the energetics of nucleotide addition to the timed release of promoter contacts. Results: A mutant that aborts less during initial transcription releases the promoter at longer RNA lengths. Conclusion: Hybrid growth remodels the protein to disrupt promoter contacts; the protein, in turn, pushes back on the hybrid, leading to abortive instability. Significance: The model extends to multisubunit RNA polymerases. RNA polymerases undergo substantial structural and functional changes in transitioning from sequence-specific initial transcription to stable and relatively sequence-independent elongation. Initially, transcribing complexes are characteristically unstable, yielding short abortive products on the path to elongation. However, protein mutations have been isolated in RNA polymerases that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. We demonstrate here that the P266L point mutation in T7 RNA polymerase, which shows dramatically reduced abortive cycling, also transitions to elongation later, i.e. at longer lengths of RNA. These two properties of the mutant are not necessarily coupled, but rather we propose that they both derive from a weakening of the barrier to RNA-DNA hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to achieve programmatically timed release of promoter contacts in the transition to elongation. Parallels in the multisubunit RNA polymerases are discussed.