Effects of Friedreich's ataxia (GAA)(n)·(TTC)(n) repeats on RNA synthesis and stability

Expansions of (GAA)(n) repeats within the first intron of the frataxin gene reduce its expression, resulting in a hereditary neurodegenerative disorder, Friedreich's ataxia. While it is generally believed that expanded (GAA)(n) repeats block transcription elongation, fine mechanisms responsible for gene repression are not fully understood. To follow the effects of (GAA)(n)·(TTC)(n) repeats on gene expression, we have chosen E. coli as a convenient model system. (GAA)(n)·(TTC)(n) repeats were cloned into bacterial plasmids in both orientations relative to a promoter, and their effects on transcription and RNA stability were evaluated both in vitro and in vivo. Expanded (GAA)(n) repeats in the sense strand for transcription caused a significant decrease in the mRNA levels in vitro and in vivo. This decrease was likely due to the tardiness of the RNA polymerase within expanded (GAA)(n) runs but was not accompanied by the enzyme's dissociation and premature transcription termination. Unexpectedly, positioning of normal- and carrier-size (TTC)(n) repeats into the sense strand for transcription led to the appearance of RNA transcripts that were truncated within those repetitive runs in vivo. We have determined that these RNA truncations are consistent with cleavage of the full-sized mRNAs at (UUC)(n) runs by the E. coli degradosome

Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy

Atomic force microscopy (AFM) has been used to image, at single molecule resolution, transcription events by Escherichia coli RNA polymerase (RNAP) on a linear DNA template with two convergently aligned λ(pr) promoters. For the first time experimentally, the outcome of collision events during convergent transcription by two identical RNAP has been studied. Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC). This discontinuous time-course enables subsequent analysis of collision events where both RNAP remain bound on the DNA. After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template. The final positions of the two RNAP indicate that these are collisions between an EC and a stalled EC (SEC) or OPC (previously referred to as sitting-ducks). Interestingly, the distances between the two RNAP show that they are not always at closest approach after ‘collision’ has caused their arrest

Non-canonical DNA transcription enzymes and the conservation of two-barrel RNA polymerases

DNA transcription depends on multimeric RNA polymerases that are exceptionally conserved in all cellular organisms, with an active site region of >500 amino acids mainly harboured by their Rpb1 and Rpb2 subunits. Together with the distantly related eukaryotic RNA-dependent polymerases involved in gene silencing, they form a monophyletic family of ribonucleotide polymerases with a similarly organized active site region based on two double-Ψ barrels. Recent viral and phage genome sequencing have added a surprising variety of putative nucleotide polymerases to this protein family. These proteins have highly divergent subunit composition and amino acid sequences, but always contain eight invariant amino acids forming a universally conserved catalytic site shared by all members of the two-barrel protein family. Moreover, the highly conserved ‘funnel’ and ‘switch 2’ components of the active site region are shared by all putative DNA-dependent RNA polymerases and may thus determine their capacity to transcribe double-stranded DNA templates

Transcriptional control in the prereplicative phase of T4 development

Control of transcription is crucial for correct gene expression and orderly development. For many years, bacteriophage T4 has provided a simple model system to investigate mechanisms that regulate this process. Development of T4 requires the transcription of early, middle and late RNAs. Because T4 does not encode its own RNA polymerase, it must redirect the polymerase of its host, E. coli, to the correct class of genes at the correct time. T4 accomplishes this through the action of phage-encoded factors. Here I review recent studies investigating the transcription of T4 prereplicative genes, which are expressed as early and middle transcripts. Early RNAs are generated immediately after infection from T4 promoters that contain excellent recognition sequences for host polymerase. Consequently, the early promoters compete extremely well with host promoters for the available polymerase. T4 early promoter activity is further enhanced by the action of the T4 Alt protein, a component of the phage head that is injected into E. coli along with the phage DNA. Alt modifies Arg265 on one of the two α subunits of RNA polymerase. Although work with host promoters predicts that this modification should decrease promoter activity, transcription from some T4 early promoters increases when RNA polymerase is modified by Alt. Transcription of T4 middle genes begins about 1 minute after infection and proceeds by two pathways: 1) extension of early transcripts into downstream middle genes and 2) activation of T4 middle promoters through a process called sigma appropriation. In this activation, the T4 co-activator AsiA binds to Region 4 of σ70, the specificity subunit of RNA polymerase. This binding dramatically remodels this portion of σ70, which then allows the T4 activator MotA to also interact with σ70. In addition, AsiA restructuring of σ70 prevents Region 4 from forming its normal contacts with the -35 region of promoter DNA, which in turn allows MotA to interact with its DNA binding site, a MotA box, centered at the -30 region of middle promoter DNA. T4 sigma appropriation reveals how a specific domain within RNA polymerase can be remolded and then exploited to alter promoter specificity

Efficient three dimensional building model generation from two dimensional floor plans

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2008.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references (p. 87).3D building models are beneficial to architects, interior designers, and ordinary people in visualizing indoor space in three dimensions. 3D building models appear to be more aesthetic to ordinary people than architectural drawings. Architects can benefit from such models in detecting any inconsistencies in their designs. This thesis describes the design and implementation of an efficient 3D building model generator (3dGen) that can automatically create 3D building models from AutoCAD drawings. This thesis explains how 3dGen takes floor plan data in XML format (generated from AutoCAD drawings), extrudes the walls and vertical surfaces and adds additional 3D information to the existing floor plan. In doing so 3dGen aims to satisfy the complete watertight space and the manifold properties and attempts to minimize the amount of 3D data by eliminating redundant geometric primitives. This thesis explains the algorithms that were employed in order to generate correct surfaces with many types of portals in them and algorithms that detect inconsistencies in the 2D architectural drawings.by Dmitry Kashlev.M.Eng