Frankia sequences exhibiting RNA polymerase promoter activity.
Frankia are Gram-positive, filamentous bacteria capable of fixing atmospheric dinitrogen either in the free-living state or in symbiosis with a variety of woody plants. Only a few Frankia genes have been sequenced and gene expression is not well characterized. To isolate a segment of Frankia DNA that functions as an RNA polymerase promoter, fragments of Frankia strain ArI5 genomic DNA were cloned upstream of a promoterless, Vibrio harveyi luxAB cassette. Constructs were screened for luminescence in E. coli and positive clones assayed for in vitro transcription activity with a partially purified Frankia RNA polymerase extract. Primer extension analysis of in vitro transcripts produced from one clone, GLO7, identified two major transcription start sites, TSP-1 and TSP-2, 52 bp apart. Deletion analysis then localized sequences essential for promoter activity. The upstream promoter region, GLO7p1, contains sequences resembling the -35 element of a Streptomyces promoter and the -35 and -10 elements of the canonical E. coli promoter. Also within this region are two pentamers identical to sequences near the 5' end of the Frankia strain CpI1 glutamine synthetase gene. The second promoter, GLO7p2, contains a putative NtrC binding site at -145 and a possible sigma(N)-RNA polymerase recognition sequence at -14 suggesting that GLO7p2 may be a nitrogen-regulated promoter. An in vivo transcript representing an ORF of 498 aa starting 64 bp downstream of the distal transcription start, TSP-1, was detected by RT-PCR. This supports the conclusion that this DNA fragment has promoter activity in vivo as well as in vitro.
Bock
Joyce V
JV
Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA1.
Battershell
Ty
T
Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA1.
Wiggington
James
J
Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA1.
John
Theodore R
TR
Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA1.
Johnson
Jerry D
JD
Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA1.
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D012321:DNA-Directed RNA Polymerases