All six GC-motifs of the SV40 early upstream element contribute to promoter activity in vivo and in vitro

EMBO J. 1985 Dec 30;4(13B):3839-49.

Abstract

Recombinants in which the six GC-motifs (I-VI) present in the upstream element of the SV40 early promoter region have been point mutated either individually or in pairs were used to determine the possible contribution of each GC-motif to the function of the overlapping early-early and late-early SV40 promoters. GC-motif I, and to a lesser extent, GC-motifs II and III, are critical for initiation at the early-early start sites. GC-motifs IV-VI play a subsidiary role. Mutations in GC-motifs I and II do not decrease the activity of the late-early promoter, whereas mutations in the GC-motifs III-VI have a moderate effect on it. The in vivo phenotype of the GC-motif mutants can be almost fully reproduced in vitro using a nuclear extract. DNase I protection footprinting experiments using wild-type or mutated templates and nuclear extracts indicate that each GC-motif behaves principally as an independent protein-binding site, presumably for transcription factor Sp1. The effect of changing the position of the 21-bp repeat region on initiation from the early-early and late-early start sites indicates that there is little flexibility in the position in which this upstream element can efficiently activate initiation of transcription from these start sites.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cloning, Molecular
  • DNA Restriction Enzymes
  • DNA, Recombinant / analysis
  • Genes, Viral*
  • Genetic Vectors
  • HeLa Cells / metabolism
  • Humans
  • Mutation
  • Plasmids
  • Promoter Regions, Genetic*
  • Simian virus 40 / genetics*
  • Transcription, Genetic

Substances

  • DNA, Recombinant
  • DNA Restriction Enzymes

Associated data

  • GENBANK/J02400
  • GENBANK/J02402
  • GENBANK/J02403
  • GENBANK/J02406
  • GENBANK/J02407
  • GENBANK/J02408
  • GENBANK/J02409
  • GENBANK/J02410
  • GENBANK/V01380