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EC20 was labeled with CTP at three positions and immobilized on Ni 2+-chelating agarose beads. The experiment began with preparation of EC stalled at position +20 (EC20) using RNAP1 (Figure 1A). For convenience, the His-tagged enzyme is designated as 1 and non-His-tagged as 2.

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The non-tagged holoenzyme was purified from greA –/ greB – cells and is free of transcript cleavage factors ( Orlova et al., 1995). To monitor the effect of trailing ECs on roadblocked ECs, we utilized T7A1 promoter DNA and two kinds of E.coli RNAPs, one that carries a His 6 tag at the C-terminus of the β′ subunit and another without the His tag. It halts the majority of ECs 14 ± 1 bp upstream of the EcoRI site ( Pavco and Steege, 1990 Nudler et al., 1995). Taken together our in vitro and in vivo results suggest that cooperation between RNAP molecules plays an important role in rendering transcription fast and processive on protein covered (physiological) DNA.Įfficient transcription through the roadblocks depends on multiple rounds of initiationĭNA-bound EcoRI restriction endonuclease ( EcoRQ111), defective in DNA cleavage ( Wright et al., 1989), is a formidable barrier to transcription. Transcript cleavage activity is not required during this process. Using two different site-specific DNA-binding proteins with high affinity for their cognate sites, we demonstrate that, in both cases, trailing ECs rescue roadblocked ECs by ‘pushing’ them forward. In the present work, we tested the hypothesis that such a cooperation mechanism between RNAP molecules also functions in overcoming elongational roadblocks. Using a reconstituted transcription system from Esherichia coli, we demonstrated that trailing ECs reactivate leading backtracked ECs at pause or arrest sites by translocating them forward ( Epshtein and Nudler, 2003). Recently, we showed that trailing ECs directly stimulate the activity of leading ECs. The elongation phase of the transcription cycle often involves multiple RNAP molecules moving, one after another, along the same DNA molecule. Con sidering the common structural organization of bacterial and eukaryotic RNAP ( Ebright, 2000 Darst, 2001 Gnatt et al., 2001), it is likely that the basic anti-roadblock mechanism is also conserved in evolution. Even less clear is how various RNAPs deal with DNA-binding proteins other than histones during elongation. Although specific protein complexes, such as FACT and Elongator, have been shown to facilitate RNAP II transcription to some extent on chromatin templates in vitro ( Orphanides et al., 1998 Kim et al., 2002), it is unclear whether these factors are sufficient for rapid chromatin transcription in vivo by RNAP II, and also RNAP I and III. Remarkably, transcript cleavage factors, as well as Elongin and Mfd, are dispensable for cell viability under physiological conditions ( Archambault et al, 1992 Selby and Sancar, 1993 Orlova et al., 1995 Yamazaki et al., 2003), suggesting that a more general transcriptional anti-arrest and anti-roadblock mechanism exists. Elongin Aso et al., 1995) or reactivate the backtracked EC by actively translocating it forward (Mfd Park et al., 2002). More specific elongation factors have been also described that can either suppress EC backtracking (e.g. It is assumed that the repeated rounds of cleavage and re-extension of RNA in blocked EC can eventually displace the roadblock, allowing transcription to proceed through the physical barrier. Cleavage of the 3′ portion of the nascent transcript reactivates the elongation complex (EC) that has been backtracked for one or more nucleotides along DNA and RNA ( Komissarova and Kashlev, 1997 Nudler et al., 1997). These factors function by inducing transcript cleavage in the RNAP catalytic site ( Izban and Luse, 1992 Borukhov et al., 1993 Rudd et al., 1994 Orlova et al., 1995). Eukaryotic and bacterial general elongation factors SII and GreA/GreB, respectively ( Reinberg and Roeder, 1987 Borukhov et al., 1993) have been shown to stimulate transcription through site-specific DNA binding proteins ( Reines and Mote, 1993 Toulmé et al., 2000). Pavco and Steege, 1990 Izban and Luse, 1991 Reines and Mote, 1993). Despite the constant presence of such nucleoprotein complexes as well as various site-specific DNA binding proteins within intragenic regions, rapid progression of elongating RNAP is not compromised in vivo, although in vitro, protein roadblocks often impede transcription (e.g. In bacteria and eukaryotes DNA is organized into a condensed structure composed, in part, of histones and histone-like proteins.

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