A transcription terminator in the groEx gene of symbiotic X-bacteria expressed in Escherichia coli.
The over-expressing groEx gene of symbiotic X-bacteria in Amoeba proteus has unique nucleotide motifs (Tx), containing two hairpins and a C-rich region at its 3'-end. To investigate the role of Tx as a transcription terminator, we mutated Tx and analyzed the effects on the expression of an upstream-located lacZ in E. coli. The level of beta-galactosidase (beta-gal) production in Tx deletion mutants was reduced to 23% of the control level. Site-directed mutation of the hairpin-1, C-rich region, and hairpin-2 reduced the beta-gal production to 28-64%, 33, and 20% of wild-type Tx, respectively. The amount of lacZ transcripts that were detected in RNA blots was proportional to the level of beta-gal. The Tx sequence had 97% termination efficiency in vivo, and the mutations in Tx resulted in read-through transcripts with significantly shortened half-life. In rho- E. coli, the level of the beta-gal production by Tx was reduced to 15% of that in rho+ E. coli. These results suggest that Tx is a Rho-dependent transcription terminator. Also, hairpin-2 is the most effective component among the three motifs of Tx for proper termination of the transcription and stability of mRNAs.
Lee
Jung Eun
JE
School of Biological Sciences, Seoul National University, Korea.
Choi
Sung Han
SH
Ahn
Tae In
TI
D000656:Amoeba / Q000382:microbiology; D000818:Animals; D001419:Bacteria / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D007763:Lac Operon; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D020871:RNA Stability; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013559:Symbiosis; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D012329:RNA, Bacterial; D001616:beta-Galactosidase