RESEARCH

Structural and functional studies of RNA polymerase and gene regulation in Methanosarcina acetivorans

Introduction:

Transcription in Archaea is similar to that in eukaryotes.  Archaea contain a eukaryotic RNA pol II-like DNA dependent RNA polymerase (RNAP) with identified homologs to 10 of the 12 subunits of the RNA polymerase molecule as well as homologs to several general transcription factors (GTF) including TBP (TATA binding protein), TFIIB (TFB in Archaea), TFIIE (TFE), and TFIIS (TFS).  There are no identified archaeal homologs to TFIIA, TFIIF, or TFIIG.  Archaea also contain a very eukaryotic-like promoter region with an identified TATA box and B recognition element (BRE) upstream of the transcription start site.  Unlike eukaryotes, Archaea contain small genomes (0.5 – 6 Mb) with a single circular chromosome.  Although histone molecules and other sequence-independent DNA binding proteins have been identified in several archaeal species, there is no strong evidence for a universal chromatin structure in Archaea.  Archaea regulate gene expression in a fashion similar to that of Bacteria by utilizing sequence-specific transcriptional repressors and activators to either compete with RNAP for the promoter region or stabilize its interaction with the promoter.

Specific Aims:

1. By what mechanism is the eukaryotic/archaeal RNA polymerase (RNAP II) catalyzing the elongation of RNA transcripts?

Without a high resolution crystal structure of RNAP (~2 Ǻ), it is not possible to discern the mechanism of RNA elongation because water molecules are not visible at lower resolutions.  The complete crystal structure of yeast RNAP II has been solved to ~3 Ǻ (too low to see water molecules).  M. acetivorans RNAP is slightly smaller than that from yeast.  Using the smaller archaeal enzyme, we hope to solve the RNAP crystal structure to ~ 2 Ǻ.   We are currently focusing on two subunits RpoE and RpoF (homologs to yeast Rpb7 and Rpb4 respectively).  In both domains, these peptides form a heterodimer that associates with the RNAP catalytic core machinery to mediate recruitment to the promoter DNA during formation of the initiation complex.  As was the case in yeast RNAP II, the RpoE/F dimer appears to impede crystallization.  If we can create RpoE/F knockout mutants in M. acetivorans, we should be able to purify the catalytic core machinery for crystallographic studies.  Until recently, this approach would not have been possible.  However, Metcalf et al., 1997 reported transformation efficiencies as high as 10⁸ transformants μg DNA^-1 10⁹ cells in M. acetivorans using liposome mediated transformation.  They have since used this method to employ homologous recombination-mediated gene replacement in the construction of proline auxotrophs.

Architecture of the 12-subunit Pol II.
4.2 Ǻ resolution ribbon model. (1)

1. What is the role of transcriptional activators in the recruitment of TFB and TBP to the promoter in M. acetivorans?

Gene regulation in Archaea like that in Bacteria is mediated by a number of sequence-specific transcriptional activators and repressors.  The roles of several transcription repressors such as Lrs14 and MDR1 have been characterized.  However, precise role of any transcriptional activators is yet to be characterized.  A number of 2D-gels generated in our lab have shown that M. acetivorans cells have markedly different protein expression profiles depending on growth substrate (in this case either acetate or methanol).  Based on these 2D-gels, we are attempting to identify at least one transcriptional activator (based on a HTH motif) that is up or down regulated on either methanol or acetate grown cells but not so on the other.  If we do discover a protein that fits these criteria, we will attempt to co-crystallize it along with the GTFs TBP and TFB, and the promoter DNA in order to determine whether or not there is a direct interaction between this transcription factor and the GTFs in the formation of the pre-initiation complex.

References:

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