In Vitro Studies of Transcript Initiation by Escherichia coli RNA Polymerase. 2. Formation and Characterization of Two Distinct Classes of Initial Transcribing Complexes

• Published in: Biochemistry (Easton) Vol. 42; no. 13; pp. 3787 - 3797
• Main Authors: Vo, Nam V, Hsu, Lilian M, Kane, Caroline M, Chamberlin, Michael J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08-04-2003
• Subjects: Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Base Sequence; Cytidine Triphosphate - chemistry; Cytidine Triphosphate - metabolism; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Escherichia coli - enzymology; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene Expression Regulation, Bacterial; Guanosine Triphosphate - chemistry; Guanosine Triphosphate - metabolism; In Vitro Techniques; Molecular Sequence Data; Nucleic Acid Heteroduplexes - metabolism; Promoter Regions, Genetic - physiology; RNA, Bacterial - metabolism; Sequence Homology, Nucleic Acid; T-Phages - genetics; Transcription Initiation Site - physiology; Transcription, Genetic; Uridine Triphosphate - chemistry; Uridine Triphosphate - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0269613

By following the kinetics of abortive and productive synthesis in single-round transcription assays, we confirm the existence of two general classes of initial transcribing complexes (ITCs), which we term “productive ITC” and “unproductive ITC”. The productive ITCs are able to escape from the promoter rapidly to produce full-length transcripts, but only after carrying out an obligate series of abortive initiation steps. The unproductive ITCs were found to synthesize mostly abortive transcripts of 2−3 nucleotides and escape from the promoter extremely slowly, if at all. Formation of the unproductive ITC is not due to the inactive RNA polymerase. Instead, RNA polymerase molecules recovered from both the productive and unproductive ITC fractions were shown to carry out abortive and productive synthesis with both the partitioning tendency and transcription kinetics similar to those of the original enzyme. Our results suggest that early transcription complexes are partitioned into the productive and unproductive ITCs most likely during the formation of open promoter complexes. The extent of partitioning varies with individual promoter sequences and is dependent on the nature and concentration of the initiating nucleotide. Thus, multiple classes of ITCs can be formed during promoter binding and transcript initiation.