Probing RNA Structure with Chemical Reagents and Enzymes

This unit provides thorough coverage of the most useful chemical and enzyme probes that can be used to examine RNA secondary and tertiary structure. Footprinting methods are presented using dimethyl sulfate, diethyl pyrocarbonate, ethylnitrosourea, kethoxal, CMCT, and nucleases. For chemical probes, both strand scission and primer extension detection protocols are included.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143733/1/cpnc0601.pd

Broadening the mission of an RNA enzyme

The “RNA World” hypothesis suggests that life developed from RNA enzymes termed ribozymes, which carry out reactions without assistance from proteins. Ribonuclease (RNase) P is one ribozyme that appears to have adapted these origins to modern cellular life by adding protein to the RNA core in order to broaden the potential functions. This RNA-protein complex plays diverse roles in processing RNA, but its best-understood reaction is pre-tRNA maturation, resulting in mature 5' ends of tRNAs. The core catalytic activity resides in the RNA subunit of almost all RNase P enzymes but broader substrate tolerance is required for recognizing not only the diverse sequences of tRNAs, but also additional cellular RNA substrates. This broader substrate tolerance is provided by the addition of protein to the RNA core and allows RNase P to selectively recognize different RNAs, and possibly ribonucleoprotein (RNP) substrates. Thus, increased protein content correlated with evolution from bacteria to eukaryotes has further enhanced substrate potential enabling the enzyme to function in a complex cellular environment. J. Cell. Biochem. 108: 1244–1251, 2009. © 2009 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/64531/1/22367_ftp.pd

Spatial organization of genes as a component of regulated expression

http://deepblue.lib.umich.edu/bitstream/2027.42/85743/1/Pai-Engelke-2010.pd

Cellular dynamics of tRNAs and their genes

http://deepblue.lib.umich.edu/bitstream/2027.42/85744/1/Hopper-Engelke-2010.pd

Interaction of tRNA transcription factors with satellite I DNA from Xenopus laevis

A cloned repeat of Xenopus laevis satellite I DNA was tested for the ability to form stable complexes with tRNA transcription factors in vitro. In template exclusion studies, the satellite I DNA competed efficiently with a tRNA gene for binding of yeast RNA polymerase III transcription factors. DNase I footprinting further showed that transcription factor TF IIIC alone bound to satellite I DNA at both the A block and B block consensus promoter sequences immediately downstream from the transcription start point. The strength and position of these associations indicate that satellite I DNA is a potential site for association of the same DNA-binding proteins that activate tRNA gene transcription.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27395/1/0000426.pd

Silencing near tRNA genes requires nuceolar localization

http://deepblue.lib.umich.edu/bitstream/2027.42/85734/1/Wang-Engelke-2005.pd

OH as an Alternate Tracer for Molecular Gas: A Study in the W5 Star-Forming Region

Tracing molecular H2 gas in the Galactic interstellar medium is complicated by the fact that diffuse, cold H2 is not detectable. The usual tracer for molecular gas is 12CO(1-0); however, questions have been posed about the universality of CO for this purpose, and evidence has suggested reservoirs of undetected “CO-dark” molecular gas. This dissertation contributes to research into the use of OH 18 cm lines as an alternate tracer for molecular gas. The focus of this dissertation is a survey of the W5 star-forming region using the Green Bank Telescope to determine the structure and quantity of molecular gas in W5, and to compare the properties of W5 to those of a quiescent region according to both tracers. Calculating column densities of OH requires knowledge of the excitation temperature of the observed molecular transi- tion. I have measured excitation temperatures of the OH 18 cm lines in W5 using two distinct methods: the traditional “expected profile” method, and a “continuum background method.” The latter yields more precise results, and demonstrates that the excitation temperature is different for the two 18 cm main lines. Results of the OH survey in W5 are then presented. In W5, the OH and CO trace a similar morphology of molecular gas, in contrast to quiescent regions which can contain CO-dark OH detections. The molecular gas mass traced by OH emission is slightly larger than that traced by CO, but the difference is not considered significant. I propose a volume density-based explanation for the presence or absence of CO-dark molecular gas, and estimate the average volume density for three regions using a diffuse cloud model. The CO-dark gas correlates with lower volume density portions of the qui- escent region, and the highest average volume density occurs in W5. These results suggest that CO-dark molecular gas primarily exists in interstellar space outside of star-forming regions, and that volume density is the primary distinction between the molecular gas in W5 and the quiescent region. I also discuss a novel method based on excitation temperatures for estimating physical conditions in molecular gas without relying on CO

Prediction and verification of mouse tRNA gene families

http://deepblue.lib.umich.edu/bitstream/2027.42/85741/1/Coughlin-Babck-Engelke-2009nihms-109486[1].pd