Flexibility in Transcription Start-Site Selection by RNA Polymerase Involves Transcription-Bubble Expansion (“Scrunching”) or Contraction (“Unscrunching”)

Abstract

RNA polymerase (RNAP) is a molecular machine that carries out a series of reactions during transcription initiation:(i) RNAP binds to promoter DNA, yielding an RNAP-promoter closed complex (RPc).(ii) RNAP unwinds ∼13 base pairs of promoter DNA surrounding the transcription start site, forming a single-stranded region (transcription bubble), and yielding an RNAP-promoter open complex (RPo).(iii) RNAP begins synthesis of an RNA product as an RNAP-promoter initial transcribing complex (RPitc).(iv) After RNAP synthesizes an RNA product ∼11 nt in length, RNAP breaks its interactions with the promoter, escapes from the promoter, and begins transcription elongation as an RNAP-DNA elongation complex (RDe).It has been known for four decades that the transcription start site can vary over a range of at least 5 bp--comprising the default start site (position +1), downstream-shifted start sites, (positions +2 and +3), and upstream-sifted start sites (positions −2 and −1)--and that the transcription start site can be re-programmed within this range by the use of appropriate ribodinucleotide primers. However, the mechanistic basis of this flexibility in transcription start-site selection has not been known.In this work, we have used magnetic-tweezers single-molecule nanomanipulation to monitor the extent of RNAP-dependent DNA unwinding in transcription initiation complexes containing ribodinucleotide primers that re-program transcription to start at downstream-shifted start sites (positions +2 or +3) or upstream-shifted start sites (positions −2 or −1). The results indicate that re-programming the transcription start site changes the transcription-bubble size: forcing a downstream-shifted start site increases transcription-bubble size, and forcing upstream-shifted start sites decreases transcription-bubble size. The results support a model in which flexibility in transcription start-site selection is a consequence of pre-initiation transcription-bubble expansion (“pre-initiation scrunching”) or pre-initiation transcription-bubble contraction (“pre-initiation unscrunching”).