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C Nüsslein

Publications and source records attributed to C Nüsslein.

6 recordsLinked to original sources
PubMed1977

Interaction of RNA polymerase with promoters from bacteriophage fd.

Replicative form DNA of bacteriophage fd, which had been fragmented with the restriction endonuclease II from Hemophilus parainfluenzae (endo R- HpaII), was reacted with Escherichia coli RNA polymerase; the resulting stable preinitiation complexes were analysed using the filter binding assay followed by gel electrophoresis. At 120mM KCL the first-order rate constants for complex decay were determined to be 10(-2)-10(-6)s-1. The second-order rate constants for complex formation were found to be about 10(6) -10(7) M-1 s-1. From these values association constants for the individual promoters were calculated to be 2 x 10(-8) -2 x 10(-11) M-1. The rate of formation and the stability of promoter complexes was enhanced in superhelical DNA. No evidence was found for stable promoter-specific closed complexes consisting of enzyme and helical DNA. This and the kinetic data suggest that the unwinding of base pairs is already important early in promoter selection, and not only for the formation of the final open complex. The initiation of RNA synthesis form the preinitiation complex was faster than complex dissociation and complex formation for all promoters. Consequently, the initiation efficiency of a promoter is determined by the rate of complex formation, and not by its 'affinity' for the enzyme. No correlation was found between the relative order of the fd promoters for the binding and the dissociation reaction. This is explained by different structural determinants, for the two reactions, which are located in different parts of the promoter DNA.

Base Sequence↗
PubMed1975

Stabilization of promoter complexes with a single ribonucleoside triphosphate.

Under specific binding conditions RNA polymerase forms complexes at several sites of the replicative form DNA of bacteriophage fd. One of these complexes becomes stable to both high salt and low temperature after incubation with GTP. None of the complexes is stabilized by ATP. The stabilization by GTP results from the synthesis of an oligo(G) chain, which is bound in the complex. Size and pyrimidine fingerprints of the DNA segment protected by the enzyme against digestion with DNase are not changed upon initiation of oligo(G) synthesis. This result indicates that binding site and initiation site are identical parts of a promoter region.

Binding Sites↗
PubMed1975

Initiation of transcription within an RNA-polymerase binding site.

1. The 5'-terminal sequence of the RNA transcribed from bacteriophage fd replicative form DNA under the control of promotor region I has been determined to be ppp(Gp)nUpApApApGpApCpCpUpGpApUpUp. . . 2. This sequence is complementary to the 5'-terminal sequence of the minus strand of the corresponding RNA polymerase binding site I, the starting point for RNA synthesis lying approximately in the middle of the binding site. 3. This initial sequence is also transcribed faithfully from isolated complexes of RNA polymerase and binding site I, obtained by DNase digestion of complexes between RNA polymerase and fd replicative form DNA. These highly stable complexes can not be reconstituted from binding site and enzyme. 4. It is concluded that RNA polymerase binding site and initiation site are identical parts of a promoter region, and that no "drift" between these sites is required as a step in RNA chain initiation. An additional non-transcribed outside region is implicated as essential for full promoter function.

Base Sequence↗