Ribosomal RNA antitermination in vitro: requirement for Nus factors and one or more unidentified cellular components.
Using an in vitro transcription assay, we have successfully demonstrated read through of a Rho-dependent terminator by the ribosomal RNA antitermination system. The assay used a DNA template containing a promoter-antiterminator-terminator arrangement, RNA polymerase, termination factor Rho, antitermination factors NusA, NusB, NusE, and NusG, and a cellular extract depleted of NusB. Terminator read-through was highly efficient only in the presence of the extract and Nus factors, suggesting that an as yet uncharacterized cellular component is required for ribosomal antitermination. The NusB-depleted extract had no activity in the absence of NusB, confirming an absolute requirement for this protein in ribosomal RNA antitermination. The DNA template requirements were the same as those previously established in vivo; transcription of a wild-type boxA sequence is both necessary and sufficient to promote RNA polymerase modification into a terminator-resistant form.
Squires
C L
CL
Department of Biological Sciences, Columbia University, New York, NY 10027.
Greenblatt
J
J
Li
J
J
Condon
C
C
Squires
C L
CL
D001426:Bacterial Proteins / Q000494:pharmacology; D001483:Base Sequence; D002474:Cell-Free System; D004275:DNA, Ribosomal / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000494:pharmacology; D012234:Rho Factor / Q000494:pharmacology; D012269:Ribosomal Proteins / Q000494:pharmacology; D013728:Terminator Regions, Genetic / Q000235:genetics*; D014157:Transcription Factors / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*; D035602:Transcriptional Elongation Factors
D001426:Bacterial Proteins; D004275:DNA, Ribosomal; D029968:Escherichia coli Proteins; C074952:NusB protein, E coli; C070291:NusG protein, E coli; D010445:Peptide Elongation Factors; D012234:Rho Factor; D012269:Ribosomal Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; C029537:ribosomal protein S10