Despite the similarities between RNA polymerases and the common requirement for TBP, the Pol II and Pol III transcription machineries are mechanistically distinct. Pol II core promoters consists of TATA, initiator, and downstream elements that are recognized by the basal transcription machinery that contains TBP, Pol II, and general transcription factors . Upon initiation, Pol II dissociates from these general factors and associates with ����elongation factors���� that travel down the mRNA coding region . In vivo, efficient transcription requires activator proteins that bind specifically to regulatory DNA sequences and, via co-activators, stimulate the basal transcription machinery . Some Pol II-transcribed genes are regulated by repressors that bind to specific DNA sequences. The identity, quality, and location of regulatory sequences are gene-specific, with the consequence that every gene has a distinct pattern of expression. For the vast Z-VAD-FMK Caspase inhibitor majority of Pol III-transcribed genes, promoter recognition elements are located internally within the RNA coding region, and Pol III transcription involves a multi-step assembly of general initiation factors . In general, the six-subunit TFIIIC binds to the A- and B-boxes, and it acts as an assembly factor directing binding of the TBP complex, TFIIIB, to a position just upstream of the initiation site. Transcription of 5S rRNA genes requires an additional factor, TFIIIA, that binds to the Abox, C-box, and IE-element. Once TFIIIB is assembled, the RNA polymerase is recruited and directs multiple rounds of transcription. Although Pol III genes are not individually regulated in the manner of Pol II genes, they are collectively subject to the negative regulator Maf1, which inhibits transcription in response to stress signals such as oxidative stress, cell wall stress, DNA damage, or nutrient limitation . After transcription, specific nucleosides in PLX-4720 citations primary tRNA transcripts become modified to yield a mature functional tRNA . In S. cerevisiae, the initiator methionine tRNA tRNAMet i _ _ contains a unique 29-O-ribosyl phosphate modification ) at position 64 that is important for the discrimination between translational initiation and elongation . Rit1, the initiator 29-O-ribosyl phosphate transferase, is not required for normal cell growth, but a synergistic growth defect is observed in a rit1 deletion strain also containing a reduced number of initiator methionine tRNA genes . Using a genetic screen based on synthetic lethality in a rit1 mutant background to identify genes important for Pol III transcription, we have isolated mutations in the IWR1 gene.

741 proteins were associated with DNA-binding folds. Of the remaining 1121 ORFs that could not be modeled at this time, 600 are annotated as conserved hypotheticals, while another 314 are associated with cell wall processes. A sequence analysis based prediction of trans-membrane helices , indicated that 850 proteins contained at-least one membraneassociated segment. Of these, structural models are available for 251, many of which participate in cell-wall processes. Further, of the membrane associated proteins, the cytochrome C Abmole Perifosine oxidase subunitI-like fold occurs most often , followed by glycerol-3-phosphate transporter and SNF like folds, . Among the least commonly occurring folds, 9 folds are associated with extracellular processes such as toxin-anti toxin system and that are implicated in cell adhesion. Functional annotation has been carried out by broadly three different approaches: first through known fold-function associations, second through identification of known sub-structural motifs and third through binding site identification, comparison with sites of known ligands and a subsequent ligand association. Fold to function assignments have been possible for 2832 different proteins, using the classification scheme reported earlier . Seven broad functional categories associated to 50 different functions have been linked to the models of TB. Validation for this part of the annotation pipeline has been carried out on the 312 protein test set. Binding site prediction, comparison and ligand association computed using PocketDepth and PocketMatch were compared with that in their corresponding crystal structures , which indicated that ligand associations and hence annotations were in most cases correct, when the detected similarity was atleast moderately high . Similarly a comparison between the fold-based function annotation and the GO terms, also indicated broad agreement . While the annotations and a broad functional PLX-4720 chemical information category were available in the databases for many proteins based on literature and sequence analyses, several new associations have been possible through modeling and structural analyses, some examples are described later. Of the 639 conserved proteins annotated as hypothetical proteins in the TB genome, 560 proteins can now be associated with fold-based function annotation through the pipeline reported here. While 282 of these 639 proteins have an association with one or more functional categories of COG, our protocol has provided an annotation for 560 proteins through structural analysis.