Electron microscopic studies of the binding of Escherichia coli RNA polymerase to DNA. I. Characterization of the non-specific interactions of holoenzyme with a restriction fragment of bacteriophage T7 DNA

Non-specific interactions between a 3800 base-pair restriction fragment of bacteriophage T7 DNA (MboI-C) and Escherichia coli RNA polymerase holoenzyme have been examined by electron microscopy. Holoenzyme displays a relatively weak and rapidly reversible binding to DNA that is only slightly reduced at elevated salt concentrations. As the concentration of NaCl is increased from 50 mm to 200 mm, the binding constant decreases from 2 × 10⁴m^?1to 4 × 10³m^?1. It is concluded that only 1 to 2 sodium ions are released from the DNA when holoenzyme binds non-specifically.The validity of the electron microscopic technique for determining binding constants has been investigated by varying aspects of the grid surface and by examining the non-specific interactions of lac repressor with DNA.     

The kinetics of oxidation of hemerythrin species—linear free energy relationships

The second-order rate constants (M^?1sec^?1, 25°C, pH 8.2, I = 0.15 M) for the oxidation to (semi-met)₀of deoxyhemerythrin from Phascolopsis gouldii (P.g.) and Themiste zostericola (T.z.) have been determined for Fe(CN)₅(4-NH₂py)^2? (3.6 × 10⁴ T.z.,2.8 × 10²P.g.),Fe(CN)₅NH₃^2?(2.4 × 10⁴ T.z.), Fe(CN)₆^3? (1.0 × 10⁵ T.z.,1.4 × 10²P.g.), Fe(CN)₅PPh₃^2? (7.3 × 10⁵T.z.), and Fe(CN)₄dipy- (~6 × 10⁶ T.z.,7.5 × 10⁴ P.g.). Corresponding rate constants for the oxidation of (semi-met)_R to met are: Fe(CN)₅(4-NH₂py)^2? (1.2 × 10³ P.g.), Fe(CN)₆^3? (3.4 × 10⁵ T.z., 4.5 × 10 Fe(CN)₅PPh₃^2? (4.4 × 10⁴P.g.), Fe(CN)₄dipy^? (1.7 × 10⁵P.g.), and Coterpy₂³⁺ (5.1 P.g.) The rates of oxidation of deoxy- and (semi-met)_R myohemetythrin by Fe(CN)₆^3? were too rapid for stopped-flow measurement. The Marcus relationship for cross-reactions was successfully applied to these data.     

Electron microscopic studies of the binding of Escherichia coli RNA polymerase to DNA: II. Formation of multiple promoter-like complexes at non-promoter sites

The interactions between Escherichia coli RNA polymerase holoenzyme and a 3800 base-pair restriction fragment of bacteriophage T7 DNA (Mbo-IC) have been examined by electron microscopy. In addition to exhibiting weak, non-specific interactions (K_a ～ 10⁴ M^?1), RNA polymerase is able to form up to 15 to 20 relatively stable complexes with this template (K_a est 10⁹ M^?1). Only one of these complexes is formed at the T7 promoter E, that maps at 92.2 ± 1% on the conventionalgenome. The remaining complexes seem to be situated non-randomly on this fragment and possibly involve interactions with specific DNA sequences. The association kinetics of formation have been examined and give rise to a second-order rate constant of ～ 10⁵ M^?1s^?1. Formation of these complexes is markedly reduced at low temperatures. Under standard binding conditions (50 mM-NaCl) the dissociation rate of these complexes is slow ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; ~\; 30\; </mtext><mtext>min</mtext>}}$), but increases rapidly with increasing salt concentration and at reduced temperatures. It is unaffected by the presence of heparin up to 5 μg/ml. Thus it appears that E. coli RNA polymerase can form complexes with promoter-like properties at many different sites on T7 DNA.     

Genetic structure and regulation of a replicon of plasmid lambdadv.

Strains of Saccharomyces cerevisiae carrying a small double-stranded RNA species (the killer plasmid) secrete a toxin which is lethal only to strains not carrying this plasmid.We have isolated mutants in eight chromosomal genes essential for replication or maintenance of the killer plasmid, called mak1 through mak8. Seven of these genes have been mapped. mak4 and mak5 are on chromosome II; mak1 and mak8 are on chromosome XV; mak3 and mak6 are on chromosome XVI; and mak7 is on chromosome VIII. We have not yet located mak2. Two other chromosomal genes, m and pets, have been shown to be required for replication or maintenance of the killer plasmid.One allele of mak1 results in temperature sensitivity for host growth. Two independent pets isolates also result in the petite phenotype, as well as temperature sensitivity for growth.Wild-type killer strains have been reported to carry two species of doublestranded RNA of 2.5 × 10⁶ and 1.4 × 10⁶ molecular weight (designated L and M, respectively); wild-type non-killers carried only L. We estimate the size of the L and M species at 3.0 × 10⁶ and 1.7 × 10⁶ daltons, respectively. We have also detected a third species of double-stranded RNA of molecular weight 3.8 × 10⁶ (XL) present in all killer and non-killer strains examined.Mutation of any of mak1 through mak8 results in loss of the killer-associated species of double-stranded RNA (M; 1.7 × 10⁶). These mutants retain both the L species (3.0 × 10⁶) and the XL species (3.8 × 10⁶) of double-stranded RNA, and have acquired two new minor RNA species.     

Quantitation of the accumulation of histone messenger RNA during oogenesis in Xenopus leavis

The accumulation of messenger RNA coding for histone H3 in oogenesis of Xenopus laevis was studied by quantitative hybridization techniques, using a cloned genomic DNA fragment as a probe. This probe was isolated from cloned Xenopus histone DNA and contains most of the H3 coding sequences. Histone H3 mRNA accumulation was found to be completed before the maximum lampbrush stage. Hybridization of RNA blots with DNA probes containing genes for histones H2A, H2B, and H4 suggests the same accumulation pattern for the mRNAs coding for these histones as for histone H3 mRNA. The amount of H3 mRNA in the mature oocyte was established to be 130 ± 68 pg, i.e., about 5 × 10⁸ copies.     

The genetic behaviour of a cloned mouse ribosomal DNA segment mimics mouse ribosomal gene evolution.

A 1.7 × 10³ base-pair SalI fragment of mouse ribosomal gene spacer undergoes recA-independent deletions of DNA in units of approximately 126 base-pairs when cloned in λ or bacterial plasmids. When we examined the structure of the 1.7 × 10³ base-pair piece with PvuII we found it to be composed of about equal numbers of copies of each of two subrepeating units, 120 and 130 base-pairs in size. The correlation between the size of the structural subunits and the functional genetic unit of this fragment as expressed in Escherichia coli led us to study the organization of these sequences in mice. SalI (or HindII) digests of DNA samples from wild and inbred strains revealed extensive heterogeneity in the size of fragments homologous to this 1.7 × 10³ base-pair piece. A total of 15 different size classes were detected in our samples. We found that these fragments were also organized in PvuII repeating units about equal in size to the PvuII repeats in the cloned 1.7 × 10³ base-pair piece. Using an objective analytical procedure (see the Appendix) we determined that the 15 different fragments found in our mouse DNA samples probably originated as a result of genetic events based on a 135 base-pair structural unit.We consider the similarity between the size of the PvuII structural unit and the unit of genetic behavior in both the cloned and uncloned DNA samples to be significant. We suspect that there are aspects of the nucleotide structure or organization of the PvuII repeating units that play a dominant role in its genetic behavior, regardless of whether these sequences are present in E. coli or mice. We believe that the clones containing this mouse sequence may provide an experimental system for studying the nature of the genetic events that are involved in multigene evolution.     

Cloning of genes for bacteriophage T5 stable RNAs

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage λ XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo ³²P-labeled T5 4–5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI¹440 fragment contains the gene for tRNA 10 (tRNA^Asp), the EcoRI/HindIII¹220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNA^His), and the EcoRI/HindIII¹960 fragment contains only a part of the gene for tRNA 9 (tRNA^Gln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNA^Gln-tRNA^His-RNA III-RNA I-…-tRNA^Asp.