Rho-dependent transcription termination. Characterization of the requirement for cytidine in the nascent transcript.
By substituting template segments encoding AU-rich, GU-rich, and CA-rich transcripts for natural sequences upstream of the phage lambda rho-dependent tR1 termination site, we demonstrate that cytidines are required in the upstream RNA for rho-dependent termination to occur. These results are extended through in vitro mutagenesis of a template encoding an inactive AU-rich upstream sequence: certain mutant templates encoding new cytidines are able to activate rho-dependent termination. Cytidines must be dispersed over a region of the transcript in order for rho to be activated, although no specific pattern of cytidines appears to be required. The results show that no local clustering or regular spacing of cytidines is necessary and that cytidines are not used as a "ruler" to determine the location of termination sites. Rho is somewhat sensitive to the relative positions of cytidines since slightly different nascent transcripts activate rho termination activity to various degrees. A model is presented in which hexameric rho binds 78 nucleotides of contiguous RNA in a primary site, such that each monomer interacts with at least one cytidine somewhere in the 13 nucleotides allotted to the monomeric primary site.
Hart
C M
CM
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Roberts
J W
JW
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D003562:Cytidine; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D009876:Operon; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D015183:Restriction Mapping; D012234:Rho Factor / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D012329:RNA, Bacterial; D012367:RNA, Viral; D012234:Rho Factor; D003562:Cytidine; D012321:DNA-Directed RNA Polymerases