Further characterization of bacteriophage T3-induced ribonucleic acid polymerase. Studies on the size of in vitro transcripts and interaction of T3 RNA polymerase with T3 DNA

Chakraborty, P.R.; Salvo, R.A.; Majumder, H.K.; Maitra, U.

Journal of Biological Chemistry 252(18): 6485-6493

1977


ISSN/ISBN: 0021-9258
PMID: 330532
Accession: 068526411

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Abstract
The size of RNA chains synthesized in vitro by T3 RNA polymerase from a T3 DNA template was analyzed by polyacrylamide gel electrophoresis under native and denaturing conditions. In vitro, 8 discrete size classes of T3 RNA were observed, designated I-VIII. The apparent MW, estimated from their electrophoretic mobilities, are about 6.2, 4.7, 4.0, 2.8, 1.8, 0.9, 0.52 and 0.21 .times. 106, respectively. The late mRNA isolated from T3-infected Escherichia coli consist of 7 discrete size classes of RNA chains ranging in MW between 0.52-3.5 .times. 106. The largest in vitro transcripts, I, II, III and IV, and the smallest species, VIII, do not seem to have an in vivo counterpart. The remaining 3 in vitro transcripts (V, VI and VII) each appear to migrate in the gel with an apparent molecular weight similar to an in vivo RNA species. All the major in vitro T3 RNA species are initiated simultaneously and independently with GTP at the 5' terminus and are elongated at the rate of about 290 nucleotides/s at 37.degree. C under standard in vitro conditions. Except for species I and IV, all other in vitro RNA species are synthesized in approximately equimolar amounts; species I and IV approximated 1/3 and 1/2, respectively, of the molar amount of each of the other 6 species. The nature of the binary complexes formed between the phage T3 RNA polymerase and T3 DNA were studied. In the absence of all 4 nucleoside triphosphates, and at or below 0.5 mM Mg2+, T3 RNA polymerase forms a measurable complex with T3 [3H]DNA detectable by both Millipore filtration and by glycerol gradient centrifugation techniques. Upon addition of all 4 nucleoside triphosphates, the polymerase.cntdot.DNA complex initiates synthesis of RNA chains wihout the addition of any exogenous DNA. Complex formation is highly sensitive to Mg2+ and high monovalent salt concentrations; complex formation is virtually abolished at 2 mM or higher Mg2+ concentration or at 20 mM or higher K+ concentrations. Studies on the stability of the complexes between T3 DNA and T3 RNA polymerase indicate that such complexes are relatively unstable compared to complexes formed with E. coli RNA polymerase and T7 DNA; the half-time of dissociation of T3 RNA polymerase.cntdot.T3 DNA complexes at 37.degree. C is about 1 min, a value several orders of magnitude lower than complexes formed by E. coli RNA polymerase with T7 DNA.