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Transcription termination within the Escherichia coli origin of DNA replication, oriC

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Summary

Initiation of DNA replication from the Escherichia coli origin, oriC, is dependent on an RNA polymerase-mediated transcription event. The function of this RNA synthetic event in initiation, however, remains obscure. Since control of the synthesis of this RNA could serve a key role in the overall initiation process, transcription regulatory sites within and near oriC were identified using the galK fusion vector system. Our results confirm the existence of a transcription termination signal within oriC, first identified by Hansen et al. (1981), for the 16 kd transcript that is transcribed counterclockwise towards oriC. Termination is shown to be 92% efficient. A similar approach led to the detection of transcription termination within the chromosomal replication origin of Klebsiella pneumoniae. Approximately 50% of the E. coli 16 kd transcripts appear to terminate before reaching oriC between the XhoI (+416 bp) and the HindIII (+243 bp) sites. The predominant 3′ ends of RNA that enter oriC, as determined by SI nuclease mapping, were located at positions +20±2, +23±2, +37, +39, +52, +66, +92, and +107. These termination sites, which map close to RNA·DNA junctions identified by Kohara et al. (1985), appear as triplets and quadruplets. The E. coli oriC Pori-L promoter described in in vitro transcription studies by Lother and Messer (1981) was not detected in this study in either wildtype cells or isogenic dnaA mutants at the nonpermissive temperature. A new promoter activity, Pori-R1, was identified within the E. coli origin in the clockwise direction.

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Abbreviations

kb:

kilobase pairs

bp:

base pairs

kd:

kilodalton

Ap:

ampicillin

DTT:

dithiothreitol

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Author notes

  1. David E. Junker Jr.

    Present address: Syntro Corporation, 10655 Sorrento Valley Rd., 92121, San Diego, CA, USA

  2. Donna Ganea

    Present address: Behring Diagnostics, 10933 Torrey Pines Rd., 92037, La Jolla, CA, USA

Authors and Affiliations

  1. Biology Department and Molecular Biology Institute, San Diego State University, 92182, San Diego, CA, USA

    David E. Junker Jr., Luis A. Rokeach, Donna Ganea, Anne Chiaramello & Judith W. Zyskind

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  1. David E. Junker Jr.
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  2. Luis A. Rokeach
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  3. Donna Ganea
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  4. Anne Chiaramello
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  5. Judith W. Zyskind
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Communicated by D.F. Grindley

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Junker, D.E., Rokeach, L.A., Ganea, D. et al. Transcription termination within the Escherichia coli origin of DNA replication, oriC . Mol Gen Genet 203, 101–109 (1986). https://doi.org/10.1007/BF00330390

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  • DOI: https://doi.org/10.1007/BF00330390

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