Archaeal intrinsic transcription termination in vivo.

Abstract:

: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 beta-glycosidase-encoding reporter gene (TK1761). Expression of the reporter gene was almost fully inhibited by the upstream presence of 5'-TTTTTTTT (T(8)) and was reduced >70% by archaeal intergenic sequences that contained oligo(T) sequences. An archaeal intergenic sequence (t(mcrA)) that conforms to the bacterial intrinsic terminator motif reduced TK1761 expression approximately 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 (A(8)) 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.

journal_name

J Bacteriol

journal_title

Journal of bacteriology

authors

Santangelo TJ,Cubonová L,Skinner KM,Reeve JN

doi

10.1128/JB.00982-09

subject

Has Abstract

pub_date

2009-11-01 00:00:00

pages

7102-8

issue

22

eissn

0021-9193

issn

1098-5530

pii

JB.00982-09

journal_volume

191

pub_type

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