x - ray crystallographic studies on multisubunit rna polymerases ( rnaps ) from eukaryotes , bacteria and archaea have revealed highly related enzymes with structurally conserved active sites . for eukaryotic and bacterial enzymes , crystal structures of transcribing complexes have been solved , showing the locations of the dna template , the nascent rna product and the substrate - binding site . structural studies have their limitations , however , which makes a comprehensive functional screen of amino - acid substitutions in domains critical for polymerase action published in journal of biology especially useful . the results of the study by robert weinzierl 's group ( tan et al . ) are in keeping with previous structural work and will be a valuable resource for interpreting future structural , single molecule and molecular modeling experiments . initial structural studies on the prokaryotic and eukaryotic enzymes identified a conserved alpha - helical domain , termed the bridge helix , that spans between two main lobes of the enzyme ( for a useful bibliography of the structural literature on rna polymerase , see tan et al . ) . further studies revealed interactions of this helix with the dna template , distorting its path adjacent to the nucleotide - addition site . moreover , the bridge helix appeared in two separate conformations  ' straight ' in a eukaryotic rnap structure and ' kinked ' in a bacterial rnap structure  raising the possibility that the conformational dynamics of the bridge might directly control enzyme translocation : that is , bending of the bridge helix might accompany movement of the polymerase along the dna template . lately , attention has turned from the bridge helix to an adjacent domain of the enzyme , the trigger loop ( figure 1 ) . this loop is mobile or unstructured in many rnap crystal structures and appears conformationally flexible . in recent structures of transcribing complexes , the trigger loop has been seen to interact directly with template - specified nucleotide substrates , suggesting critical roles in catalysis and substrate selection . indeed , mutations in trigger - loop residues alter elongation rate , transcriptional pausing , response to regulators , substrate selection and transcriptional fidelity . the loop makes extensive contacts with the bridge helix , also raising the possibility that the concerted actions of these two domains may underlie key rnap dynamics during the nucleotide - addition cycle . rna polymerase ii elongation complex showing positions of bridge helix ( bh ) and trigger loop ( tl ) . ( a ) cartoon representation of 10-subunit pol ii elongation complex ( pdb : 2e2h )   with protein shown in charcoal gray , template dna in blue , rna product in red , and non - template dna in green . buried within , near the active site , are the tl ( cyan ) and bh ( magenta ) . ( b ) transparent view of ( a ) showing the position of the bh and tl relative to the nucleic acid scaffold within pol ii . ( c ) rotated view of ( b ) showing the position of the bh and tl relative to the 3 ' end of the rna product ( the site of transcript elongation , shown by an arrow ) . structural studies have important limitations in regard to understanding the transcription mechanism at the molecular level . first , transcribing complexes designed for crystallographic studies are unavoidably compromised by alterations made to prevent phosphodiester bond formation . in some structural studies of eukaryotic rnaps , as with those of numerous dna polymerases , putative reaction intermediates contain 3'-deoxynucleotide - terminated nucleic acid primers to prevent chain elongation . in other eukaryotic rnap structures , and in a structure of a bacterial rnap transcribing complex , non - hydrolyzable substrates were used to prevent phosphodiester bond formation . in either case a second limitation of crystal structures is that they only provide snapshots of stable conformations , failing to fully reveal the dynamics involved in catalysis of phosphodiester bond formation and translocation of rnap along the template . current structures of transcribing rnaps have not attained atomic resolution , placing the reaction mechanism out of reach . indeed , as modeled , eukaryotic rnap   and bacterial elongation complexes   containing bound substrates differ in putatively critical interactions between trigger loop and nucleotide that could have profound consequences for the rnap catalytic mechanism . in light of these shortcomings , biochemical studies such as those of tan the current study by tan et al . uses a highly automated , high - throughput approach for the production and characterization of rnap variants developed previously by weinzierl and colleagues . their approach takes advantage of a completely recombinant system for production of the rnap from the archaeon methanocaldococcus jannaschii . this recombinant archaeal polymerase is composed of nine subunits , a ' , a " , b ' , b " , d , h , l , n and p ( the other three subunits of the native enzyme showed no detectable effect on function in vitro and were therefore omitted ) . recombinant rnaps may be expressed , purified , assembled and characterized in batches of 96 rnap variants over 12 days , making large - scale analysis of systematically designed rnap substitution mutants feasible . in addition , the approach is highly automated and can probably be implemented for the study of any number of enzymatic systems , thus making it a generally useful technology for protein - engineering studies . in the ' rna polymerase factory ' , all amino - acid substitutions at a number of positions can be evaluated in parallel , giving an essentially unbiased approach to structure - function analysis that is not necessarily based on pre - existing structural information or limited by numbers of mutants that can be evaluated . such high - throughput evaluation does not have to rely on in vivo genetic identification of variants , which can be limited by methods of mutagenesis that are able to generate only subsets of all possible substitutions , or by traditional site - directed mutagenesis approaches that rely on prior rationales for the design of variants . in previous proof - of - principle work , weinzierl and colleagues applied their automated analysis to all 19 amino - acid substitution variants for m. jannaschii rnap subunit a ' ( mja ' ) residue g825 , within the bridge helix domain . in their current study , they extend this analysis to an astonishing 323 variants within the bridge helix , comprising all 19 single amino - acid substitutions within a 17 amino - acid stretch of the helix . the authors also characterize an additional 38 substitutions , encompassing all 19 single amino - acid substitutions of trigger - loop residues mja " g72 and mja " i98 . in regard to elucidating the interactions between bridge helix and trigger loop , it is fascinating that the authors identify a large number of super - activating substitutions within the bridge helix as well as some within the trigger loop . it was already known that certain trigger - loop substitutions in bacterial rnap and yeast rna polymerase ii ( pol ii ) allow the enzymes to transcribe more quickly than wild - type variants . in the study by tan et al . gain - of - function substitutions are present within the region observed to bend in crystal structures and along one side of the helix facing the trigger loop . double - substitution mutants containing gain - of - function substitutions in both bridge helix and trigger loop have no greater gain of activity than the most severe single substitution , suggesting that the two domains function together to promote transcription . as mentioned above , the bridge helix can be observed in a number of conformations in crystal structures , with a kink or a bend in various positions ( figure 2 ) . in structures of thermus thermophilus ( tth ) movement of the trigger loop towards the bridge helix in a saccharomyces cerevisiae rnap also appears to perturb the helix by altering its path in the direction of the observed kink , although only to a small extent . in each case , the bend is in the same direction away from the trigger loop . a kinked bridge helix has not yet been observed in a structure of the tth rnap elongation complex , raising the possibility that an extreme bridge helix kink may not necessarily function in transcription elongation . the relative flexibility of the bridge helix , perhaps not unexpected for such an isolated helix , is underscored by the results of tan et al . , who show that the bridge helix can be somewhat tolerant to helix - breaking prolyl substitutions along its length , including one substitution at the position of the observed helix kink ( mja ' s824p ) that super - activates rnap . furthermore , kinked bridge helix structures reveal an interaction between two residues of the helix ( analogs of mja ' residues 823 and 829 ) that may stabilize the kinked conformation . substitutions at either of these putatively kink - stabilizing positions compromise rnap activity to various extents . notably , re - establishment of a possible interaction between the two substituted positions results in a moderate restoration of activity , consistent with an on - pathway role for the observed interaction . these results suggest that the kinked bridge helix promotes rnap activity , but whether in catalysis or translocation remains to be determined . conformations of the bridge helix ( bh ) observed in crystal structures of multisubunit rnaps . ( a ) kinked orientation of the bh in some tth rnap crystal structures . bh from tth holoenzyme without nucleic acids in the presence of streptolydigin [ pdb:1zyr ] ( yellow ) . ( b ) straight orientation of the bh in tth elongation complex structures with or without nucleoside triphosphate ( ntp ) substrate . bh from the tth transcribing complex without ntp substrate ( the trigger loop ( tl ) in this structure is in the ' out ' position away from the substrate - addition site ) [ pdb:2o5i ] ( orange ) . bh from tth elongation complex with ntp substrate ( the tl is folded ' in ' and contacting the ntp substrate in this structure ) [ pdb : id 2o5j ] ( lime green ) . ( c ) mild perturbation of the pol ii bh in elongation complex with matched ntp substrate in position for addition . bh from s. cerevisiae ( sce ) pol ii elongation complex without substrate , pdb 1i6h ( magenta ) . bh from the sce pol ii elongation complex with mismatched substrate ( the tl is in the ' out ' position away from substrate - addition site in this structure ) [ pdb:1r9 t ] ( teal ) . bh from the sce pol ii elongation complex with matched substrate ( the tl is folded ' in ' and contacting the ntp substrate in this structure ) [ pdb:2e2h ] ( blue ) . the rna polymerase factory has proved its usefulness for the characterization of a large number of rnap variants . the next stage of this approach is likely to include comprehensive analysis of trigger - loop residues and active - site residues predicted to contact nucleotide substrates . a subset of the mutants identified by tan et al . should be characterized with more traditional assays that are able to directly measure rnap elongation rate , substrate selection and propensity for pausing . information on such a wide spectrum of mutants will benefit single - molecule studies in which rnap variants with specific defects can be used to probe models of the transcription mechanism . in addition , molecular modeling studies that aim to accurately describe and elucidate the rnap mechanism can be tested for recapitulation of phenotypes demonstrated for a wide range of rnap variants , such as those described by tan et al . finally , biophysical studies able to directly probe and characterize rnap active - site dynamics , such as bridge helix and trigger loop movement , and how such movement is impacted by substrate binding and perturbation of rnap structure are likely to be important for understanding of the rnap mechanism at the near - atomic level . an eventual complete elucidation of the rnap mechanism , specifically , the identification of the entire retinue of rnap amino acids required for catalysis and translocation , will be a first step for understanding the regulation of rnap activity in vivo . we acknowledge funding from the nih to rdk ( gm36659 and gm49985 ) . due to journal policy , we have only sparingly referenced the literature and apologize to those whose work we were unable to specifically reference .