Effect of a low-molecular-weight DNA binding protein, H1 factor, on the in vitro transcription of the lactose operon in Escherichia coli.
H1 protein, a heat-stable low-molecular-weight DNA-binding factor previously described by Cukier-Kahn et al. [Proc. Nat. Acad. Sci USA (1972) 69, 3643-3647] markedly stimulates in vitro synthesis of lac-specific RNA directed by bacteriophage lambdah80 dlac or phi80 dlac DNA templates in the presence of purified E. coli RNA polymerase holoenzyme. The extent of stimulation obtained by addition of H1 alone is usually greater than that observed with the cAMP receptor protein-cAMP combination. H1 effect varies quite appreciably (from 4- to 16-fold) with the functional state of the promoter, being much larger with lambdah80 dlac p-s, a transducing DNA carrying a superpromoter mutation, than with lambdah80 dlac p+. H1 and cAMP receptor protein effects are nearly additive, although interpretation of the data obtained at high H1 concentration is complicated by the appearance of some inhibitory property. While the cAMP-receptor-protein-mediated synthesis is asymmetrical ("I" strand almost exclusively copied), the degree of asymmetry observed with H1 is less pronounced, suggesting asymmetrical copying from the lac promoter and symmetric transcription from other regions of the DNA. Synthesis of lac-specific RNA from lambdah80 dtrp/lac or phi80 dlac p-r uv5 templates, in which lac promoters are insensitive to cAMP receptor protein, either as a result of lac fusion to the trp operon or mutation in the lac promoter, is totally H1-insensitive. Glycerol (10-15% w/w) can fully substitute for H1 in stimulating lac RNA synthesis in a fashion analogous to that reported for the cAMP receptor protein-cAMP system. The possibility that H1 acts by causing conformational modifications at the promoter level in a way that increases its functional state, and that this effect is more pronounced with operons sensitive to cAMP receptor protein, is discussed.
D001426:Bacterial Proteins / Q000378:metabolism*; D003090:Coliphages / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004267:DNA Viruses / Q000378:metabolism; D004269:DNA, Bacterial / Q000096:biosynthesis*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005809:Genes, Regulator; D066298:In Vitro Techniques; D007785:Lactose / Q000378:metabolism*; D008970:Molecular Weight; D009154:Mutation; D009690:Nucleic Acid Conformation; D009876:Operon; D011485:Protein Binding; D012967:Sodium Dodecyl Sulfate; D014158:Transcription, Genetic; D014161:Transduction, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012967:Sodium Dodecyl Sulfate; D000242:Cyclic AMP; D012321:DNA-Directed RNA Polymerases; D007785:Lactose