Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1983
The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to t... more The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to the 1821st nucleotide upstream from the beginning of the sequence coding for mature 16 S rRNA. In vitro transcription experiments indicated the presence of two new promoters in this region, located more than 1 kb upstream from the known P1 and P2 promoters of rrnB. Previous electron microscopic studies demonstrated that these sites bind RNA-polymerase very strongly. In vitro transcription, starting at these sites reads through the entire region into the rrnB gene without termination. A similar uninterrupted transcription into rrnB in vivo can be demonstrated by S1-mapping, and by fusing the DNA containing the new promoters (but not P1 and P2) to the lacZ gene. Thus it seems likely that these promoters (P3 and P4) belong functionally to the rrnB gene and play some role in its regulation of expression.
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1980
The promoter region of two bacterial rRNA genes was investigated by electron-microscopic analysis... more The promoter region of two bacterial rRNA genes was investigated by electron-microscopic analysis of polymerase binding, transcription initiation and nitrocellulose filtration of RNA-polymerase-DNA complexes, using restriction endonuclease generated fragments of recombinant plasmids and a transducing phage. The following observations have been made: 1. Two transcription initiation sites have been located approximately 200 and 300 base pairs upstream from the beginning of the sequence coding for mature 16 S rRNA. 2. Polymerase binding at these sites can be observed electronmicroscopically and a 360 base-pair fragment containing these sites binds to nitrocellulose in the presence of RNA-polymerase. This complex dissociates even at moderately high (0.1-0.2 M) salt concentrations. Although transcription initiation is reported to be more frequent at the first of these sites, the binding is much stronger at the second site. 3. In the case of the rrnD gene, BamHI cleaves a few base pairs upstream from the first transcription start site. This cleavage destroys polymerase binding at this site but does not influence binding at the second site. 4. At higher polymerase/DNA ratio four weak but distinct and regularly spaced binding sites can be observed preceding the two initiation sites at approximately 1000, 820, 640 and 440 base pairs before the mature 16 S rRNA sequence. 5. An extremely strong binding site is located about 1300 base pairs upstream from the beginning of the 16 S rRNA sequence. Very little (if any) initiation occurs at this site. The possibility is discussed that the noninitiating binding sites preceding the two transcription start points might functionally belong to the promoter region.
In vitro transcription of ribosomal RNA was studied on the DNA of the transducing bacteriphage la... more In vitro transcription of ribosomal RNA was studied on the DNA of the transducing bacteriphage lambdarifd 18, which carries an rRNA transcription unit from Escherichia coli. rRNA synthesis was preferential at all polymerase/DNA ratios tested, and at the optimal 0.3 weight ratio nearly 60% of the transcript was rRNA. At this ratio the principal product of transcription comigrated in acrylamide-agarose electrophoresis with authentic 30-S rRNA precursor synthesized in vivo. In the presence of rifampicin more than one equivalent of rRNA was synthesized, thus suggesting the existence of two initiation sites for rRNA on the phage DNA. Similar results were obtained on E. coli DNA. Preincubation with heparin virtually eliminated the transcription of rRNA, in sharp contrast with the results of similar experiments on bacterial DNA where rRNA genes were transcribed 4--5 times in the presence of heparin. The possible explanation of this difference between rRNA promotors on the phage and the bacterial DNA are discussed.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1983
The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to t... more The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to the 1821st nucleotide upstream from the beginning of the sequence coding for mature 16 S rRNA. In vitro transcription experiments indicated the presence of two new promoters in this region, located more than 1 kb upstream from the known P1 and P2 promoters of rrnB. Previous electron microscopic studies demonstrated that these sites bind RNA-polymerase very strongly. In vitro transcription, starting at these sites reads through the entire region into the rrnB gene without termination. A similar uninterrupted transcription into rrnB in vivo can be demonstrated by S1-mapping, and by fusing the DNA containing the new promoters (but not P1 and P2) to the lacZ gene. Thus it seems likely that these promoters (P3 and P4) belong functionally to the rrnB gene and play some role in its regulation of expression.
Bacterial ribosomal RNA synthesis was studied in an in vitro system in which the presence of hepa... more Bacterial ribosomal RNA synthesis was studied in an in vitro system in which the presence of heparin prevented reinitiation of transcription. The number of heparin-resistant binary complexes of RNA-polymerase and E. coli DNA depended strongly on the quality of the template. High-molecular weight DNA was a much superior template than DNA prepared by conventional techniques. Using this high-molecular weight DNA as template the amount of ribosomal RNA synthetized in one round of transcription was found to be 4-5 fold higher than the amount of rDNA present. Controls have shown that the transcription probably started at the proper initiation sites and no significant read-through form distant promoters contributed to this effect. If the binary polymerase-DNA complexes were dissociated in the presence of 0.5 M KC1 prior to transcription all RNA synthesis was strongly reduced but the proportion of rRNA increased in the transcript. However, in this case the amount of rRNA did not exceed the amount of rDNA. We propose that the promoters of the rRNA genes are complex structures, able to store 4-5 molecules of RNA polymerase and of these several polymerase only one is bound in an extremely salt-resistant form.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1983
The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to t... more The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to the 1821st nucleotide upstream from the beginning of the sequence coding for mature 16 S rRNA. In vitro transcription experiments indicated the presence of two new promoters in this region, located more than 1 kb upstream from the known P1 and P2 promoters of rrnB. Previous electron microscopic studies demonstrated that these sites bind RNA-polymerase very strongly. In vitro transcription, starting at these sites reads through the entire region into the rrnB gene without termination. A similar uninterrupted transcription into rrnB in vivo can be demonstrated by S1-mapping, and by fusing the DNA containing the new promoters (but not P1 and P2) to the lacZ gene. Thus it seems likely that these promoters (P3 and P4) belong functionally to the rrnB gene and play some role in its regulation of expression.
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1980
The promoter region of two bacterial rRNA genes was investigated by electron-microscopic analysis... more The promoter region of two bacterial rRNA genes was investigated by electron-microscopic analysis of polymerase binding, transcription initiation and nitrocellulose filtration of RNA-polymerase-DNA complexes, using restriction endonuclease generated fragments of recombinant plasmids and a transducing phage. The following observations have been made: 1. Two transcription initiation sites have been located approximately 200 and 300 base pairs upstream from the beginning of the sequence coding for mature 16 S rRNA. 2. Polymerase binding at these sites can be observed electronmicroscopically and a 360 base-pair fragment containing these sites binds to nitrocellulose in the presence of RNA-polymerase. This complex dissociates even at moderately high (0.1-0.2 M) salt concentrations. Although transcription initiation is reported to be more frequent at the first of these sites, the binding is much stronger at the second site. 3. In the case of the rrnD gene, BamHI cleaves a few base pairs upstream from the first transcription start site. This cleavage destroys polymerase binding at this site but does not influence binding at the second site. 4. At higher polymerase/DNA ratio four weak but distinct and regularly spaced binding sites can be observed preceding the two initiation sites at approximately 1000, 820, 640 and 440 base pairs before the mature 16 S rRNA sequence. 5. An extremely strong binding site is located about 1300 base pairs upstream from the beginning of the 16 S rRNA sequence. Very little (if any) initiation occurs at this site. The possibility is discussed that the noninitiating binding sites preceding the two transcription start points might functionally belong to the promoter region.
In vitro transcription of ribosomal RNA was studied on the DNA of the transducing bacteriphage la... more In vitro transcription of ribosomal RNA was studied on the DNA of the transducing bacteriphage lambdarifd 18, which carries an rRNA transcription unit from Escherichia coli. rRNA synthesis was preferential at all polymerase/DNA ratios tested, and at the optimal 0.3 weight ratio nearly 60% of the transcript was rRNA. At this ratio the principal product of transcription comigrated in acrylamide-agarose electrophoresis with authentic 30-S rRNA precursor synthesized in vivo. In the presence of rifampicin more than one equivalent of rRNA was synthesized, thus suggesting the existence of two initiation sites for rRNA on the phage DNA. Similar results were obtained on E. coli DNA. Preincubation with heparin virtually eliminated the transcription of rRNA, in sharp contrast with the results of similar experiments on bacterial DNA where rRNA genes were transcribed 4--5 times in the presence of heparin. The possible explanation of this difference between rRNA promotors on the phage and the bacterial DNA are discussed.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1983
The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to t... more The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to the 1821st nucleotide upstream from the beginning of the sequence coding for mature 16 S rRNA. In vitro transcription experiments indicated the presence of two new promoters in this region, located more than 1 kb upstream from the known P1 and P2 promoters of rrnB. Previous electron microscopic studies demonstrated that these sites bind RNA-polymerase very strongly. In vitro transcription, starting at these sites reads through the entire region into the rrnB gene without termination. A similar uninterrupted transcription into rrnB in vivo can be demonstrated by S1-mapping, and by fusing the DNA containing the new promoters (but not P1 and P2) to the lacZ gene. Thus it seems likely that these promoters (P3 and P4) belong functionally to the rrnB gene and play some role in its regulation of expression.
Bacterial ribosomal RNA synthesis was studied in an in vitro system in which the presence of hepa... more Bacterial ribosomal RNA synthesis was studied in an in vitro system in which the presence of heparin prevented reinitiation of transcription. The number of heparin-resistant binary complexes of RNA-polymerase and E. coli DNA depended strongly on the quality of the template. High-molecular weight DNA was a much superior template than DNA prepared by conventional techniques. Using this high-molecular weight DNA as template the amount of ribosomal RNA synthetized in one round of transcription was found to be 4-5 fold higher than the amount of rDNA present. Controls have shown that the transcription probably started at the proper initiation sites and no significant read-through form distant promoters contributed to this effect. If the binary polymerase-DNA complexes were dissociated in the presence of 0.5 M KC1 prior to transcription all RNA synthesis was strongly reduced but the proportion of rRNA increased in the transcript. However, in this case the amount of rRNA did not exceed the amount of rDNA. We propose that the promoters of the rRNA genes are complex structures, able to store 4-5 molecules of RNA polymerase and of these several polymerase only one is bound in an extremely salt-resistant form.
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