Howard Gamper

A topological model for transcription based on unwinding angle analysis of E. coli RNA polymerase binary, initiation and ternary complexes

DNA unwinding induced by Escherichia coli RNA polymerase is measured for binary, initiation and ternary complexes formed from a unique promoter sequence on simian virus 40 DNA. At 37 degrees C the complexes all have an unwinding angle of 17 +/- 1 base pairs (580 degrees +/- 30 degrees). This unwinding is attributed to an enzyme-stabilized separation of the double helix at the promoter site, which is maintained throughout initiation and elongation. There is no heterogeneity in the unwinding angle of the ternary complex as it progresses down the helical template. The constant DNA unwinding during all phases of transcription leads us to propose the existence of unwindase and rewindase activities on the enzyme that allow it to travel down the helix like a nut on a DNA bolt. During elongation, the unwindase unwinds the DNA helix while the rewindase, lagging by 17 base pairs, displaces the RNA transcript and reseals the helix. Both activities induce a rotation in the DNA double helix relative to the polymerase. The RNA-DNA hybrid also rotates, maintaining both ends of that helix fixed relative to the catalytic and windase sites. Formation of an RNA-DNA hybrid which spans the distal end of the DNA unwound region is proposed as a possible mechanism for polymerase pausing and termination. This model requires that the polymerase direct the transcript past the noncoding DNA strand. Pausing occurs 16-20 nucleotides downstream from the centers of appropriately sized dyad symmetry elements.

EFFICIENT FORMATION OF A CROSSLINKABLE HMT MONOADDUCT AT THE Kpn I RECOGNITION SITE

Abstract The double‐stranded Kpn I linker, CGGTACCG, is readily crosslinked by 4′‐hydroxymethyl‐4,5′,8‐trimethylpsoralen (HMT). Under identical conditions, the corresponding Bam HI linker, CGGATCCG, is resistant to modification. The differential reactivity of HMT towards the two sequences is also observed with SV40 DNA where the Kpn I restriction site is ten‐fold more susceptible to HMT modification than the Bam HI site. Selective reaction with the Kpn I site is attributed to preferential intercalation of HMT into the TpA sequence. The availability of crosslinked Kpn I linker has allowed us to characterize the kinetics of photoreversal. Upon irradiation at 254 nm, the diadduct reverses at a faster rate than the monoadduct, leading to an accumulation of the latter. The resultant monoadduct reforms crosslink rapidly upon irradiation at 365 nm. DNA oligomers like the Kpn I linker, which can be modified by the above protocol to contain a crosslinkable HMT monoadduct, could he exploited as photoreactive sequence‐specific probes.

Solution hybridization of crosslinkable DNA oligonucleotides to bacteriophage M13 DNA: Effect of secondary structure on hybridization kinetics and equilibria☆

Several DNA oligonucleotides have been photochemically modified with the furocoumarin 4-hydroxymethyl-4,5,8-trimethylpsoralen (HMT) such that each contained a single HMT furan side monoadduct to thymidine at a unique 5 TpA 3 sequence. When these oligonucleotides were hybridized to their respective complements, the HMT adduct could be driven to form an interstrand crosslink by irradiation of the hybrid with 360 nm light. The ability to crosslink probe-target complexes has allowed us to determine the kinetics and the extent of hybridization in solution between these oligonucleotides and their complementary sequences in single-stranded bacteriophage M13 DNA. Our data indicate that these parameters are strongly influenced by the existence of local as well as global secondary structure in the viral DNA. During hybridization, rearrangement of this secondary structure so as to expose the target sequence can be rate-limiting. Upon attainment of equilibrium, only a portion of the target sequence may be hybridized to the probe with the remainder involved in intrastrand base-pairing. Using crosslinkable oligonucleotide probes hybridized and irradiated near the melting temperature of the respective probe-target complex one can partially overcome these secondary structure effects.

Interaction of Escherichia coli RNA polymerase with DNA in an elongation complex arrested at a specific psoralen crosslink site

We have probed the interaction of Escherichia coli RNA polymerase with DNA in an elongation complex arrested by a site-specifically placed psoralen crosslink using DNase I footprinting techniques. The psoralen derivative 4-hydroxymethyl-4,5,8-trimethylpsoralen was first placed at a specific site in the middle of a chemically synthesized double-stranded DNA fragment containing an E. coli RNA polymerase promoter at one end. The psoralen molecule was photochemically attached to two adjacent thymidine residues on opposite strands as a diadduct. Using this crosslinked DNA as the template for transcription, we found that the E. coli RNA polymerase was blocked at the psoralen diadduct, yielding a transcript 29 nucleotides long. The arrested elongation complex inhibited DNase I digestion of both the coding strand and the non-coding strand from about 22 nucleotides upstream to 15 nucleotides downstream from the diadduct. These results, which suggest that the unwindase and the catalytic sites of the polymerase are very close to each other, have been incorporated into a model of the transcription elongation complex.

Enzyme organization in the proline biosynthetic pathway of Escherichia coli.

Abstract The conversion of glutamic acid to proline by an Escherichia coli extract was studied. The activity was dependent upon the presence of ATP and NADPH and was largely unaffected by the presence of NH 3 or imidazole. The first two pathway enzymes appear to exist as a complex which stabilizes a labile intermediate postulated as γ-glutamyl phosphate. Attempted synthesis of this compound was unsuccessful due to its spontaneous cyclization to 2-pyrrolidone 5-carboxylate. Dissociation of the enzyme complex upon dilution of the extract is presumed responsible for an experimentally observed “dilution effect”. E. coli pro A − and pro B − auxotroph extracts failed to complement one another in the biosynthesis of proline. This is attributed to the lack of a dynamic equilibrium between the complex and its constituent enzymes. In vivo studies with E. coli showed no evidence for metabolic channeling in the final reaction of proline synthesis, the reduction of Δ 1 -pyrroline 5-carboxylate.

Biochemical basis for the acquisition of resistance to benzo[a]pyrene in clones of mouse liver cells in culture☆

In a Namru mouse liver epithelial cell strain designated NMuLi, aryl hydrocarbon hydroxylase (AHH) activity peaked at 12 h post-induction with 1 microgram/ml of benzo(a)pyrene (BaP) in both confluent and growing cells. Maximal levels of AHH activity were reached on day two post-plating. This induced activity was inhibited in vitro 78% by gassing the incubation mixture with carbon monoxide for 15 s, and inhibited 93% by addition of 40 microgram/ml of 7,8 benzoflavone(BF). Induced AHH levels were higher in epithelial clones that were sensitive to the toxicity of BaP than in resistant clones. The survival fraction of clones from NMuLi and of subclones derived from a sensitive clone of NMuLi after BaP treatment was a negative exponential function of the maximal induced AHH activity in the clones. One of the clones, NMuLi cl 8, was extremely susceptible to the toxic effects of BaP, the +/-(trans)-7alpha, 8beta-dihydroxy-7,8-dihydro-BaP(7,8-diol), and the (+/-)-7alpha, 8beta-dihydroxy-9beta, 10beta-epoxy-7,8,9,10-tetrahydro-BaP (diol-epoxide), known metabolites of BaP. The toxicity of BaP and the 7,8 diol to this clone was inhibited by BF, suggesting that these cells possessed an enzyme activity inhibitable by BF that could epoxidize BaP to the 7,8 oxide and then epoxidize the resultant 7,8 diol to the diol-epoxide. Another clone derived from NMuLi, clone 7, was relatively resistant to the toxic effects of BaP and the 7,8-diol, but still extremely susceptible to the toxic effects of the diol-epoxide. The slight toxicity to BaP in this clone was inhibited by BF, but the toxicity of the 7,8-diol to this clone was not inhibited by BF. A typical cytochrome P450 inhibitor, metyrapone, had no effect on the toxicity of BaP, the 7,8-diol, or the diol-epoxide to either clone 7 or clone 8. The results suggest that these liver cells possess two enzymes that play some role in polycyclic hydrocarbon-induced toxicity. Enzyme A, a BaP-inducible enzyme that is inhibitable by BF, efficiently metabolizes BaP to the 7,8-diol and the 7,8-diol to the diol-epoxide. It is responsible for most of the hydrocarbon toxicity. Enzyme B is not inhibitable by BF and metabolizes the 7,8-diol less efficiently to the diol-epoxide or efficiently to other, less toxic products.

Base specificity in the binding of benzo[a]pyrene diol epoxide to simian virus 40 DNA

[14C]Simian virus 40 (SV40) DNA was reacted with [3H]7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to give 0.60 adducts per genome. The modified DNA was digested to completion with Hind III restriction endonuclease and the 6 fragments isolated by polyacrylamide gel electrophoresis. Hydrocarbon binding to the fragments was proportional to their guanine--cytosine (G--C) content, reflecting selective reaction of the hydrocarbon with deoxyguanosine residues. No sites unusually susceptible to alkylation were detected.