Abstract
Universally conserved factors from NusG family bind at the upstream fork junction of transcription elongation complexes and modulate RNA synthesis in response to translation, processing, and folding of the nascent RNA. Escherichia coli NusG enhances transcription elongation in vitro by a poorly understood mechanism. Here we report that E. coli NusG slows Gre factor-stimulated cleavage of the nascent RNA, but does not measurably change the rates of single nucleotide addition and translocation by a non-paused RNA polymerase. We demonstrate that NusG slows RNA cleavage by inhibiting backtracking. This activity is abolished by mismatches in the upstream DNA and is independent of the gate and rudder loops, but is partially dependent on the lid loop. Our comprehensive mapping of the upstream fork junction by base analogue fluorescence and nucleic acids crosslinking suggests that NusG inhibits backtracking by stabilizing the minimal transcription bubble.
Highlights
To control when and how fast genes are expressed, the activity of RNA polymerase (RNAP) is tightly regulated
The transcription elongation complex (TEC) was assembled on a synthetic nucleic acid scaffold and contained the fully complementary transcription bubble flanked by 20-nucleotide DNA duplexes upstream and downstream (Figure 2—figure supplement 1)
Time-resolved measurements of nucleotide incorporation by assembled TEC accompanied by forward translocation and, in a separate experiment, pyrophosphorolysis of extended TEC accompanied by backward translocation in the presence and absence of saturating concentration of NusG (2 mM, see later) (Figure 2A–C)
Summary
To control when and how fast genes are expressed, the activity of RNA polymerase (RNAP) is tightly regulated. Much of transcription regulation in all domains of life takes place at the initiation stage by modulating activities of promoters. The examples of on/off regulation at the transcript elongation stage, such as promoter-proximal pauses in metazoans (Adelman and Lis, 2012) and antitermination in prokaryotes (Santangelo and Artsimovitch, 2011), are known. Transcription elongation control is mediated by coupling of transcription to downstream processes, such as RNA translation, processing, and folding (Proshkin et al, 2010; Bubunenko et al, 2013). The multisubunit RNAPs evolved to elongate relatively inefficiently in the absence of proper coupling, thereby enabling the downstream processes to control the elongation rate. The ubiquitous family of NusG proteins (SPT5/SPT4 in archaea and yeast, DSIF in mammals) are the central components which mediate coupling between transcription and the downstream processes (Werner, 2012)
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