Analysis of upstream activation of the vnfH promoter of Azotobacter vinelandii

Umesh K. Bageshwar, Ramesh Raina, Nirupam Roy Choudhury, H. K. Das

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

BAL-31 deletion products of the DNA fragment containing the vnfH promoter and upstream region, when cloned in a transcriptional fusion vector and analyzed for vnfH expression in Azotobacter vinelandii, revealed that the upstream activator sequence of the vnfH promoter lies about 140 nucleotides upstream of the promoter. Subsequent substitution and deletion analysis by oligonucleotide-directed mutagenesis in the upstream region of the vnfH promoter showed that sequences 5'-GTACCATGCGGAAC-3' and 5'-GTACCTGCGGGTAC-3', located 170 and 140 nucleotides upstream of the vnfH promoter, respectively, are both required for vnfH expression. Addition of four nucleotides in the intervening sequence between the vnfH promoter and the putative VnfA (analog of NifA of the conventional molybdenum-dependent nitrogen-fixation pathway) binding site resulted in a drastic reduction of expression from the vnfH promoter in Azotobacter vinelandii, whereas addition of 10 nucleotides in the intervening sequence did not affect the expression. Therefore, the face of the helix-dependent contact appeared to be important. DNA bending seemed to play a crucial role in expression from vnfH promoter. The intervening sequence exhibited characteristics of sequence-dependent intrinsically curved DNA, as shown by anomalous low gel mobility with polyacrylamide gel electrophoresis, electron microscopy, and computer simulated curvature analysis. Distamycin at very low concentrations significantly reduced the anomaly in electrophoretic mobility of the intervening DNA sequence.

Original languageEnglish (US)
Pages (from-to)405-415
Number of pages11
JournalCanadian Journal of Microbiology
Volume44
Issue number5
DOIs
StatePublished - 1998
Externally publishedYes

Fingerprint

Azotobacter vinelandii
Activation Analysis
Introns
Nucleotides
Chemical activation
Genetic Promoter Regions
DNA
Nitrogen fixation
Dimercaprol
Electrophoretic mobility
Nitrogen Fixation
Mutagenesis
Molybdenum
DNA sequences
Site-Directed Mutagenesis
Electrophoresis
Oligonucleotides
Electron microscopy
Polyacrylamide Gel Electrophoresis
Electron Microscopy

Keywords

  • Azotobacter vinelandii
  • DNA bending
  • Promoter-lacZ fusion
  • vnfA
  • vnfH

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Immunology
  • Microbiology

Cite this

Analysis of upstream activation of the vnfH promoter of Azotobacter vinelandii. / Bageshwar, Umesh K.; Raina, Ramesh; Choudhury, Nirupam Roy; Das, H. K.

In: Canadian Journal of Microbiology, Vol. 44, No. 5, 1998, p. 405-415.

Research output: Contribution to journalArticle

Bageshwar, Umesh K. ; Raina, Ramesh ; Choudhury, Nirupam Roy ; Das, H. K. / Analysis of upstream activation of the vnfH promoter of Azotobacter vinelandii. In: Canadian Journal of Microbiology. 1998 ; Vol. 44, No. 5. pp. 405-415.
@article{9472340047ef4af091c3877eac187558,
title = "Analysis of upstream activation of the vnfH promoter of Azotobacter vinelandii",
abstract = "BAL-31 deletion products of the DNA fragment containing the vnfH promoter and upstream region, when cloned in a transcriptional fusion vector and analyzed for vnfH expression in Azotobacter vinelandii, revealed that the upstream activator sequence of the vnfH promoter lies about 140 nucleotides upstream of the promoter. Subsequent substitution and deletion analysis by oligonucleotide-directed mutagenesis in the upstream region of the vnfH promoter showed that sequences 5'-GTACCATGCGGAAC-3' and 5'-GTACCTGCGGGTAC-3', located 170 and 140 nucleotides upstream of the vnfH promoter, respectively, are both required for vnfH expression. Addition of four nucleotides in the intervening sequence between the vnfH promoter and the putative VnfA (analog of NifA of the conventional molybdenum-dependent nitrogen-fixation pathway) binding site resulted in a drastic reduction of expression from the vnfH promoter in Azotobacter vinelandii, whereas addition of 10 nucleotides in the intervening sequence did not affect the expression. Therefore, the face of the helix-dependent contact appeared to be important. DNA bending seemed to play a crucial role in expression from vnfH promoter. The intervening sequence exhibited characteristics of sequence-dependent intrinsically curved DNA, as shown by anomalous low gel mobility with polyacrylamide gel electrophoresis, electron microscopy, and computer simulated curvature analysis. Distamycin at very low concentrations significantly reduced the anomaly in electrophoretic mobility of the intervening DNA sequence.",
keywords = "Azotobacter vinelandii, DNA bending, Promoter-lacZ fusion, vnfA, vnfH",
author = "Bageshwar, {Umesh K.} and Ramesh Raina and Choudhury, {Nirupam Roy} and Das, {H. K.}",
year = "1998",
doi = "10.1139/cjm-44-5-405",
language = "English (US)",
volume = "44",
pages = "405--415",
journal = "Canadian Journal of Microbiology",
issn = "0008-4166",
publisher = "National Research Council of Canada",
number = "5",

}

TY - JOUR

T1 - Analysis of upstream activation of the vnfH promoter of Azotobacter vinelandii

AU - Bageshwar, Umesh K.

AU - Raina, Ramesh

AU - Choudhury, Nirupam Roy

AU - Das, H. K.

PY - 1998

Y1 - 1998

N2 - BAL-31 deletion products of the DNA fragment containing the vnfH promoter and upstream region, when cloned in a transcriptional fusion vector and analyzed for vnfH expression in Azotobacter vinelandii, revealed that the upstream activator sequence of the vnfH promoter lies about 140 nucleotides upstream of the promoter. Subsequent substitution and deletion analysis by oligonucleotide-directed mutagenesis in the upstream region of the vnfH promoter showed that sequences 5'-GTACCATGCGGAAC-3' and 5'-GTACCTGCGGGTAC-3', located 170 and 140 nucleotides upstream of the vnfH promoter, respectively, are both required for vnfH expression. Addition of four nucleotides in the intervening sequence between the vnfH promoter and the putative VnfA (analog of NifA of the conventional molybdenum-dependent nitrogen-fixation pathway) binding site resulted in a drastic reduction of expression from the vnfH promoter in Azotobacter vinelandii, whereas addition of 10 nucleotides in the intervening sequence did not affect the expression. Therefore, the face of the helix-dependent contact appeared to be important. DNA bending seemed to play a crucial role in expression from vnfH promoter. The intervening sequence exhibited characteristics of sequence-dependent intrinsically curved DNA, as shown by anomalous low gel mobility with polyacrylamide gel electrophoresis, electron microscopy, and computer simulated curvature analysis. Distamycin at very low concentrations significantly reduced the anomaly in electrophoretic mobility of the intervening DNA sequence.

AB - BAL-31 deletion products of the DNA fragment containing the vnfH promoter and upstream region, when cloned in a transcriptional fusion vector and analyzed for vnfH expression in Azotobacter vinelandii, revealed that the upstream activator sequence of the vnfH promoter lies about 140 nucleotides upstream of the promoter. Subsequent substitution and deletion analysis by oligonucleotide-directed mutagenesis in the upstream region of the vnfH promoter showed that sequences 5'-GTACCATGCGGAAC-3' and 5'-GTACCTGCGGGTAC-3', located 170 and 140 nucleotides upstream of the vnfH promoter, respectively, are both required for vnfH expression. Addition of four nucleotides in the intervening sequence between the vnfH promoter and the putative VnfA (analog of NifA of the conventional molybdenum-dependent nitrogen-fixation pathway) binding site resulted in a drastic reduction of expression from the vnfH promoter in Azotobacter vinelandii, whereas addition of 10 nucleotides in the intervening sequence did not affect the expression. Therefore, the face of the helix-dependent contact appeared to be important. DNA bending seemed to play a crucial role in expression from vnfH promoter. The intervening sequence exhibited characteristics of sequence-dependent intrinsically curved DNA, as shown by anomalous low gel mobility with polyacrylamide gel electrophoresis, electron microscopy, and computer simulated curvature analysis. Distamycin at very low concentrations significantly reduced the anomaly in electrophoretic mobility of the intervening DNA sequence.

KW - Azotobacter vinelandii

KW - DNA bending

KW - Promoter-lacZ fusion

KW - vnfA

KW - vnfH

UR - http://www.scopus.com/inward/record.url?scp=0031817353&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031817353&partnerID=8YFLogxK

U2 - 10.1139/cjm-44-5-405

DO - 10.1139/cjm-44-5-405

M3 - Article

C2 - 9699296

AN - SCOPUS:0031817353

VL - 44

SP - 405

EP - 415

JO - Canadian Journal of Microbiology

JF - Canadian Journal of Microbiology

SN - 0008-4166

IS - 5

ER -