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Journal of Bacteriology, November 2009, p. 7102-7108, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00982-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Archaeal Intrinsic Transcription Termination In Vivo{triangledown}

Thomas J. Santangelo, L'ubomíra Cubonová, Katherine M. Skinner, and John N. Reeve*

Department of Microbiology, Ohio State University, Columbus, Ohio 43210

Received 25 July 2009/ Accepted 31 August 2009

Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) strains have been constructed with synthetic and natural DNA sequences, predicted to function as archaeal transcription terminators, identically positioned between a constitutive promoter and a β-glycosidase-encoding reporter gene (TK1761). Expression of the reporter gene was almost fully inhibited by the upstream presence of 5'-TTTTTTTT (T8) and was reduced >70% by archaeal intergenic sequences that contained oligo(T) sequences. An archaeal intergenic sequence (tmcrA) that conforms to the bacterial intrinsic terminator motif reduced TK1761 expression ~90%, but this required only the oligo(T) trail sequence and not the inverted-repeat and loop region. Template DNAs were amplified from each T. kodakarensis strain, and transcription in vitro by T. kodakarensis RNA polymerase was terminated by sequences that reduced TK1761 expression in vivo. Termination occurred at additional sites on these linear templates, including at a 5'-AAAAAAAA (A8) sequence that did not reduce TK1761 expression in vivo. When these sequences were transcribed on supercoiled plasmid templates, termination occurred almost exclusively at oligo(T) sequences. The results provide the first in vivo experimental evidence for intrinsic termination of archaeal transcription and confirm that archaeal transcription termination is stimulated by oligo(T) sequences and is different from the RNA hairpin-dependent mechanism established for intrinsic bacterial termination.

* Corresponding author: Mailing address: Department of Microbiology, Ohio State University, Columbus, OH 43210. Phone: (614) 292-1267. Fax: (614) 292-8120. E-mail: reeve.2{at}osu.edu

{triangledown} Published ahead of print on 11 September 2009.

Journal of Bacteriology, November 2009, p. 7102-7108, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00982-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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