## 2 Pausing  
The rate of transcription elongation by _E. coli_ RNAP is not uniform. RNA synthesis is characterized by pauses, some of which may be brief and resolved spontaneously, whereas others may lead to TEC backtracking. Backtracked TEC can restart when acted upon by GreA/B factors, which restore the 3'- end of the nascent transcript to the active center. Pausing may regulate gene expression, as in the case of attenuation or phage \(\lambda\) gene Q antitermination. In this section we will discuss recent advances in understanding the mechanism and specificity of pausing. Elongation rate and pausing are determined by template sequence and RNA structure (e.g., stem-loops) and involve at least two components of the RNAP catalytic center, the bridge helix (BH) and trigger loop (TL). Elongation is proposed to occur in two steps. First, the TL folds in response to NTP binding. Mutational analyses indicate that this conformational change in the TL can be rate-limiting, and reflects the ability of the incoming NTP to bind to TEC. The second step is the incorporation of the NTP and the release of pyrophosphate. Kinetic conformational changes in the TL, however, do not account for pause recovery, since the TL remains unfolded during a pause [1]. Pausing not associated with backtracking is frequent, occurring on average every 100 bases of DNA [2]. The paused intermediates are distinct from the intermediates of the main reaction pathway, and they are not associated with translocation delay. The paused complex contains the 3'- end of the transcript in the active center and is capable of binding the next cognate NTP. It is highly dependent on the NMP at the 3'OH end of the nascent transcript. For example, pausing at C37 on a T7A1 template is significantly reduced when the template substitutes a G37 for C37. Substitutions at position 38 also affect pause times, which might indicate the ability of the incoming XTP to bind to TEC. These considerations have lead to the idea that the 3'OH base may not be fully base-paired with the template, even though it lies in the post-translocated state. [PERSON] and his coworkers [3] analyzed pauses that result from failure of TEC to translocate from the elongation-inactive pre-translocated state to the active post-translocated state. These pauses reflect the ability of RNAP core to sense the identity of base pairs at most of the positions of the RNA-DNA hybrid. It is not clear if the sequence or the structure of the hybrid induces pausing. Some of these pauses are associated with \"backstepping\", _i.e._, movement of TEC towards the promoter by one bp, with associated displacement of the 3' ribonucleotide from the active center. A genome-wide _in vivo_ analysis of TEC occupancy on the _E. coli_ chromosome defined almost 20,000 pause sites [4]. Analysis of these sites revealed a consensus sequence that consists of G-10 Y-1 G+1 (where -1 corresponds to the position of the RNA 3' end). A similar result was obtained by [PERSON] _et al._[5] This sequence is proposed to induce pausing through an interaction between RNAP core enzyme and a core recognition element (CRE) located at the 3'- end of the RNA:DNA duplex. The interaction stabilizes TEC in the pre-translocated state, thus inhibiting addition of the next nucleotide to the nascent transcript. The G-10 favors the pretranslocated state by enhancing duplex stability; each position of the consensus pause sequence is predicted to favor the pretranslocated state over the posttranslocated state (the -10G through effects on duplex stability, the -1 Y through effects on active-center binding, and the +1 G through both). Mutational probing of RNAP supports this model. Thus, RNAP BD446 hydrogen bonds with Watson-Crick atoms of G complexed with CRE, suggesting that D446 recognizes this nucleotide. As predicted, RNAP BD446A cannot distinguish G, A, T, or an abasic site at position G+1 _in vitro_ and pauses with equal efficiency on the various templates.