Factors Stabilizing DNA Folding in Bacterial Chromosomes

Pettijohn, David E.; Hecht, Ralph M.; Stonington, Oliver G.; Stamato, Thomas D.

doi:10.1007/978-94-011-6132-9_12

Cited by 35 publications

(53 citation statements)

References 20 publications

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“…As for the dimer-tRNA-DNA complex found in vitro, we do not know its role, if any, in vivo. The type of structure we have described illustrates the potential asymmetry of core enzyme oligomers, and could play a role in maintaining the structure of the folded chromosome [46,47].…”

Section: Localisation Of Core Enzyme In the Cellmentioning

confidence: 91%

On the Binding of tRNA to Escherichia coli RNA Polymerase. Interactions between the Core Enzyme, DNA and tRNA

1981

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We have investigated the interplay between the binding of tRNA and DNA to core RNA polymerase. We show that the monomer core enzyme can bind stably to either DNA or tRNA, whereas the dimer core can fix both DNA and tRNA in a stable ternary complex.We have examined the kinetics of the exchange between DNA and tRNA bound to the core enzyme. DNA bound to monomer core can be rapidly displaced by tRNA without prior dissociation of the core from the DNA. Similarly tRNA bound to the core can be displaced by DNA without prior dissociation of the tRNA. We confirm the result of Hinkle and Chamberlin [J. Mol. Biol. 70, 157-185 (1972)] that, in contrast, the core enzyme must first dissociate from one DNA molecule before it can transfer to another DNA. As this dissociation is very slow we suggest that, in vivo, the tRNA can act as a 'porter' providing the core enzyme with a more kinetically favourable path to transfer from one DNA site to another.The importance of tRNA in the regulation of RNA polymerase in Escherichia coli has been highlighted by the recent work of Travers and his collaborators [I -31 who have presented evidence that a specific tRNA, fMet-tRNAy" is capable of guiding holo RNA polymerase away from some promotors and towards others. However, aside from these specific effects, it has been known for some time that E. coli RNA polymerase has a strong general affinity for all forms Recently, we have shown that the core enzyme, particularly, forms very tight complexes with both charged and uncharged tRNA and, when complexed, exists as a mixture of monomer and dimer [9,10] which interconvert very slowly. The relative amount of monomer and dimer is not affected by the excess of tRNA. The monomer complex consists of one c12pp' assembly and one tRNA molecule whilst the dimer complex possesses one tight tRNA binding site and one site where tRNA fixation is more labile [lo]. This implies that the dimer adopts an asymmetric structure in the presence of tRNA.Up to 80% of the RNA polymerase in the cell is core enzyme [I 1,121 and at least half of this is not actively involved in transcription [I3 -151. Studies with mini cells [lb-181 and folded chromosomes [19,20] have shown that this non-transcribing enzyme is distributed between the cytoplasm and the E. coli chromosome. As the core enzyme has a very strong general affinity for DNA [21,22] can only partially displace tRNA from the core and vice vcrsu. Indeed stable ternary complexes between DNA, tRNA and the core enzyme can form [lo]. Here we show that such stable ternary complexes can form only with the dimer core enzyme and that the monomer enzyme can fix only either DNA or tRNA.The complexes between the core enzyme and DNA are very stable and have a long lifetime [21,23]. We have, therefore, investigated the kinetics of exchange of bound tRNA or DNA with free DNA or tRNA, in order to determine whether DNA-bound core enzyme can be passed more rapidly to another site on the DNA via an intermediary tRNA molecule. Our results are discussed with respect to the role of core...

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Section: Localisation Of Core Enzyme In the Cellmentioning

confidence: 91%

On the Binding of tRNA to Escherichia coli RNA Polymerase. Interactions between the Core Enzyme, DNA and tRNA

1981

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“…As revealed by the 3H:14C ratio before and after banding, the RNA:DNA ratio in the fixed nucleoids was unchanged (Table 1). Previous studies have demonstrated that most of the 3H-uridine is incorporated into RNA when nucleoids were labeled as in 10,11 3 Figure lb…”

Section: Resultsmentioning

confidence: 99%

Analysis by Isopycnic centrifugation of isolated nucleoids of Escherichia coli

1975

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The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 ± 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 ± 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid.

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“…Further work by Pettijohn et al (1973) revealed that the difference in sedimentation coefficients obtained by the t,.o groups was due to differences in the lysing temperature. Lysis at 0-4oC results in 3200S nucleoids, whereas lysis at 20°C yields nucleoids with a mean sedimentation coefficient of 1600S.…”

Section: Ultraviolet Radiation Damage and Repairmentioning

confidence: 97%

“…The more rapidly-sedimenting nucleoids were shown to be associated wi th a specific fraction of the cell membrane due to the presence of specifïc membrane proteins, lipids, and phospholipids (Pettijohn et al ~ 1973;Worcel and Burgi, 1974). Direct examination of such nucleoids in the electron microscope also revealed membrane fragments as.sociated with the DNA (Pettijohn et al~ 1973;Delius and Worcel, 1974).…”

Section: Ultraviolet Radiation Damage and Repairmentioning

confidence: 97%

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Rate zonal density gradient ultracentrifugation analysis of repair of radiation damage to the folded chromosome of Escherichia coli

Ulmer¹

1978

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Factors Stabilizing DNA Folding in Bacterial Chromosomes

Cited by 35 publications

References 20 publications

On the Binding of tRNA to Escherichia coli RNA Polymerase. Interactions between the Core Enzyme, DNA and tRNA

On the Binding of tRNA to Escherichia coli RNA Polymerase. Interactions between the Core Enzyme, DNA and tRNA

Analysis by Isopycnic centrifugation of isolated nucleoids of Escherichia coli

Rate zonal density gradient ultracentrifugation analysis of repair of radiation damage to the folded chromosome of Escherichia coli

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