Abstract
The trigger loop (TL) forms a conserved element in the RNA polymerase active centre that functions in the elongation phase of transcription. Here, we show that the TL also functions in transcription initiation and termination. Using recombinant variants of RNA polymerase from Pyrococcus furiosus and a reconstituted transcription system, we demonstrate that the TL is essential for initial RNA synthesis until a complete DNA–RNA hybrid is formed. The archaeal TL is further important for transcription fidelity during nucleotide incorporation, but not for RNA cleavage during proofreading. A conserved glutamine residue in the TL binds the 2’-OH group of the nucleoside triphosphate (NTP) to discriminate NTPs from dNTPs. The TL also prevents aberrant transcription termination at non-terminator sites.
Highlights
RNA polymerases (RNAPs) carry out transcription in all living organisms
To analyse whether the trigger loop (TL) RNAP mutants have an overall defect in promoter-dependent transcription, we subjected them to a promoter-specific transcription assay in which we used the strong P.furiosus glutamate dehydrogenase promoter in the presence of general transcription initiation factors (GTIFs) (Figure 2A)
Previous TL analysis concentrated on transcription elongation, we report here functions of the TL during transcription initiation and termination (Figure 7)
Summary
RNA polymerases (RNAPs) carry out transcription in all living organisms. Archaeal RNAP and eukaryotic RNAP II use the same core promoter elements, the TATA box and TFIIB recognition element (BRE), and interact with homologous general transcription initiation factors (GTIFs), TATAbinding protein (TBP) and transcription factor B (TF(II)B), which govern promoter DNA recognition and opening [7,10,11,12]. A third archaeal initiation factor, transcription factor E (TFE), corresponds to the N-terminal part of eukaryotic TFIIEa and interacts with the DNA non-template strand to stabilize the pre-initiation complex [3,4,7]. When the RNA reaches a critical length, RNAP dissociates from GTIFs and enters productive elongation [15,16]. The RNAP active site uses two Mg2+ ions to catalyse RNA chain growth by phosphodiester bond formation
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