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Mutations affecting RNA-DNA hybrid formation of the ColE1 replication primer RNA. Restoration of RNA I sensitivity to a copy-number mutant by second-site mutations.
J Mol Biol. 1992 Aug 20; 226(4):997-1008.JM

Abstract

Certain high copy-number mutants of the ColE1 plasmid produce a primer RNA that, unlike the wild-type, is resistant to inhibition by the plasmid-encoded replication inhibitor RNA I. We show that this resistance is associated with the ability of mutant primer RNA to hybridize to the DNA template strand more efficiently than does the wild-type transcript in vitro. We have isolated two second-site intramolecular suppressor mutations that partially restore wild-type copy number behavior to the high copy-number mutant in vivo. Each of these mutations alters a second base in primer RNA near the original mutation. We show that the primer RNA made by the pseudo-revertants regained wild-type-like sensitivity to RNA I in vitro. Also, the efficiency of RNA-DNA hybrid formation by the pseudo-revertant primer RNAs is restored to a level similar to that of wild-type primer. Using non-denaturing gel electrophoresis as an indication of RNA conformation, we identified two primer RNA conformers, each of 550 nucleotides, whose equilibrium distribution differs between wild-type and the mutant plasmid. The pseudo-revertant plasmids have a conformer distribution similar to that of wild-type, indicating that these primer sequence changes have long-range effects on primer conformation. An oligonucleotide complementary to the primer domain containing the mutation reduced hybrid formation when present during primer elongation. These results indicate that the copy-number behavior of these plasmids is a consequence of conformational alterations in primer RNA that alter its hybridization efficiency with the DNA template strand and its sensitivity to inhibition by RNA I.

Authors+Show Affiliations

Department of Biology, Indiana University, Bloomington 47405.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

1381446

Citation

Fitzwater, T, et al. "Mutations Affecting RNA-DNA Hybrid Formation of the ColE1 Replication Primer RNA. Restoration of RNA I Sensitivity to a Copy-number Mutant By Second-site Mutations." Journal of Molecular Biology, vol. 226, no. 4, 1992, pp. 997-1008.
Fitzwater T, Yang YL, Zhang XY, et al. Mutations affecting RNA-DNA hybrid formation of the ColE1 replication primer RNA. Restoration of RNA I sensitivity to a copy-number mutant by second-site mutations. J Mol Biol. 1992;226(4):997-1008.
Fitzwater, T., Yang, Y. L., Zhang, X. Y., & Polisky, B. (1992). Mutations affecting RNA-DNA hybrid formation of the ColE1 replication primer RNA. Restoration of RNA I sensitivity to a copy-number mutant by second-site mutations. Journal of Molecular Biology, 226(4), 997-1008.
Fitzwater T, et al. Mutations Affecting RNA-DNA Hybrid Formation of the ColE1 Replication Primer RNA. Restoration of RNA I Sensitivity to a Copy-number Mutant By Second-site Mutations. J Mol Biol. 1992 Aug 20;226(4):997-1008. PubMed PMID: 1381446.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mutations affecting RNA-DNA hybrid formation of the ColE1 replication primer RNA. Restoration of RNA I sensitivity to a copy-number mutant by second-site mutations. AU - Fitzwater,T, AU - Yang,Y L, AU - Zhang,X Y, AU - Polisky,B, PY - 1992/8/20/pubmed PY - 1992/8/20/medline PY - 1992/8/20/entrez SP - 997 EP - 1008 JF - Journal of molecular biology JO - J Mol Biol VL - 226 IS - 4 N2 - Certain high copy-number mutants of the ColE1 plasmid produce a primer RNA that, unlike the wild-type, is resistant to inhibition by the plasmid-encoded replication inhibitor RNA I. We show that this resistance is associated with the ability of mutant primer RNA to hybridize to the DNA template strand more efficiently than does the wild-type transcript in vitro. We have isolated two second-site intramolecular suppressor mutations that partially restore wild-type copy number behavior to the high copy-number mutant in vivo. Each of these mutations alters a second base in primer RNA near the original mutation. We show that the primer RNA made by the pseudo-revertants regained wild-type-like sensitivity to RNA I in vitro. Also, the efficiency of RNA-DNA hybrid formation by the pseudo-revertant primer RNAs is restored to a level similar to that of wild-type primer. Using non-denaturing gel electrophoresis as an indication of RNA conformation, we identified two primer RNA conformers, each of 550 nucleotides, whose equilibrium distribution differs between wild-type and the mutant plasmid. The pseudo-revertant plasmids have a conformer distribution similar to that of wild-type, indicating that these primer sequence changes have long-range effects on primer conformation. An oligonucleotide complementary to the primer domain containing the mutation reduced hybrid formation when present during primer elongation. These results indicate that the copy-number behavior of these plasmids is a consequence of conformational alterations in primer RNA that alter its hybridization efficiency with the DNA template strand and its sensitivity to inhibition by RNA I. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/1381446/Mutations_affecting_RNA_DNA_hybrid_formation_of_the_ColE1_replication_primer_RNA__Restoration_of_RNA_I_sensitivity_to_a_copy_number_mutant_by_second_site_mutations_ L2 - https://linkinghub.elsevier.com/retrieve/pii/0022-2836(92)91048-T DB - PRIME DP - Unbound Medicine ER -