Transcription termination: primary intermediates and secondary adducts.
In living organisms, stable elongation complexes of RNA polymerase dissociate at specific template positions in a process of transcription termination. It has been suggested that the dissociation is not the immediate cause of termination but is preceded by catalytic inactivation of the elongation complex. In vitro reducing ionic strength can be used to stabilize very unstable and catalytically inactive complex at the point of termination; the previous biochemical characterization of this complex has led to important conclusions regarding termination mechanism. Here we analyze in detail the complexes formed between DNA template, nascent RNA, and Escherichia coli RNA polymerase during transcription through the tR2 terminator of bacteriophage lambda. At low ionic strength, the majority of elongation complexes fall apart upon reaching the terminator. Released RNA and DNA efficiently rebind RNA polymerase (RNAP) and form binary RNAP.RNA and RNAP.DNA complexes, which are indistinguishable from binary complexes obtained by direct mixing of the purified nucleic acids and the enzyme. A small fraction of elongation complexes that reach termination point escapes dissociation because RNA polymerase has backtracked from the terminator to an upstream DNA position. Thus, transcription elongation to a terminator site produces no termination intermediates that withstand dissociation in the time scale appropriate for biochemical studies.
Kashlev
Mikhail
M
NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, USA. mkashlev@mail.ncifcrf.gov
Komissarova
Natalia
N
D010582:Bacteriophage lambda / Q000378:metabolism; D001483:Base Sequence; D004247:DNA; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D029968:Escherichia coli Proteins; D011196:Potassium Permanganate / Q000737:chemistry; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
D029968:Escherichia coli Proteins; C079667:GreB protein, E coli; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D011196:Potassium Permanganate; D004247:DNA; D012321:DNA-Directed RNA Polymerases