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.

FIG. 1.

Genome organization, sequences assayed, and reporter gene expression in T. kodakarensis strains. (A) The trpE-TK1761 cassette, its transformation, and its recombination into the genome of T. kodakarensis KW128 to generate T. kodakarensis TS372 have been described in detail (34, 35). For each of the T. kodakarensis strains constructed, the synthetic oligonucleotide given adjacent to the TS strain designation was inserted, as indicated, beginning at position +10 downstream of the site of P_hmtB-directed transcription initiation (+1). The locations of the primers (sequences are available from T.J.S.) used to generate transcription templates from genomic DNAs are indicated by small filled rectangles. The β-glycosidase activity encoded by the TK1761 gene present in each T. kodakarensis strain is shown in the bar graph as a percentage of the activity in T. kodakarensis TS372. The values are averages for triplicate assays of lysates from at least three independent cultures of each strain. (B) Sequences amplified from T. kodakarensis intergenic regions (IR) downstream from the genes listed (designated by “TK” followed by a number). Each sequence was positioned upstream of TK1761, as indicated in panel A, to generate the T. kodakarensis strain with the TS number given on the left. The β-glycosidase activity present in each T. kodakarensis strain is shown in the bar graph, as in panel A, as a percentage of the activity in T. kodakarensis TS372. (C) The t_mcrA sequence (31), positioned upstream of TK1761 in T. kodakarensis TS442, is shown with the 5′ and 3′ inverted-repeat sequences, intervening loop, and T-trail regions identified. The derivatives of t_mcrA, and the mcrA coding sequences, which were identically positioned upstream of TK1761, are given to the right of the corresponding T. kodakarensis TS strain designations. The β-glycosidase activity present in each T. kodakarensis strain is shown in the bar graph, as in panel A, as a percentage of the activity in T. kodakarensis TS372.

FIG. 2.

Template sequences and transcription termination in vitro. (A) Sequence of the P_hmtB promoter and downstream region of the linear 372 template. This template was amplified, using primers that hybridized to regions (indicated by filled bars), from T. kodakarensis TS372 genomic DNA. The TATA box, site of transcription initiation (), ribosome binding site (RBS), and 5′ sequence of TK1761 (shaded box) are identified (34, 35). The additional sequences present in templates 552, 553, 554, and 557 are listed with the T₈, A₈, and (TA)₄ sequences boxed. (B) Electrophoretic separation of the transcripts synthesized in vitro from linear (upper gel) and supercoiled, plasmid (lower gel) templates. The linear templates were generated by PCR as for panel A. The plasmid templates were the plasmids used to transform T. kodakarensis KW128 to generate T. kodakarensis strains TS372, TS552, TS553, TS554, and TS557. The [³²P]ApC-labeled transcripts were separated by electrophoresis, visualized, and quantified using a Storm 840 phosphorimager (GE Healthcare). Transcripts that were terminated from +10 on templates 372 and 557 are identified above and below the upper gel, respectively, by the nucleotides at that position in the 372 and 557 sequences (see panel A). The transcripts that were terminated within T₈, A₈, and (TA)₄ on the linear templates are boxed. The regions of the gels containing abortive, terminated, runoff (↓), or read-through () transcripts are indicated. (C) Percentages of transcripts initiated and extended beyond +10 that were terminated on linear versus circular versions of each template.

FIG. 3.

Transcription termination in vitro by t_mcrA on linear and circular templates. (A) Electrophoretic separation of the transcripts synthesized in vitro from linear templates amplified from T. kodakarensis TS372, TS442, TS443, TS444, TS445, TS555, and TS556. Templates 442, 443, and 445 have identical 5′ sequences (see Fig. 1C), and the transcripts that were terminated from +10 on these templates are identified between the upper two gels by the nucleotide at that position. Transcripts that were terminated from + 10 on templates 555 and 556 are identified above and below the lower gel, respectively, by the nucleotide at that position. Transcripts that were terminated within the T-trail and loop regions of t_mcrA are identified and boxed. (B) Electrophoretic separation of the transcripts synthesized from plasmid templates pTS372, pTS442, pTS443, pTS444, pTS445, pTS555, and pTS556. Transcripts that were terminated within T-trail sequences are boxed. The regions of the gels containing abortive, terminated, runoff (↓) (A), or read-through (, ) (B) transcripts are indicated. (C) Percentages of transcripts initiated and extended beyond +10 that were terminated on linear versus circular versions of each template.