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Structure and interactions of archaeal RNase P proteins Mth Rpp29 and Pfu Rpp21

Boomershine, William P

Abstract Details

2005, Doctor of Philosophy, Ohio State University, Ohio State Biochemistry Program.
RNase P is an essential enzyme in all three domains of life required to cleave the 5’ leader sequence of precursor tRNA (pre-tRNA) yielding the mature tRNA. The most highly studied RNase P is from Escherichia coli where the enzyme is a ribozyme containing one large (~120 kDa), catalytically active RNA subunit (M1 RNA) and one small (~15 kDa) protein cofactor (C5 protein). While M1 RNA is catalytically active in vitro in the absence of the C5 protein under high salt conditions, the C5 protein is absolutely required for activity in vivo. In contrast to bacterial RNase P, the counterparts in eukarya and archaea contain one large RNA subunit and multiple proteins. While the bacterial and human RNase P RNA subunits share some predicted secondary structural similarities, there is no sequence homology between the bacterial protein subunit to any of the human protein subunits. On the other hand, four archaeal protein subunits were identified through sequence homology to four of the human protein subunits help stabilize the active conformation of the RNA subunit in vivo subunits (hPop5, Rpp21 Rpp29 and Rpp30). Our goal is to understand the role of the protein subunits in the archaeal/eukaryal RNase P holoenzyme. We hypothesize that they interact through an induced-fit mechanism where both the protein and RNA subunits fold into the active conformation upon binding. We have decided to study archaeal RNase P instead of human RNase P for several reasons. First, archaeal RNase P can be reconstituted from in vitro transcribed RNA and recombinantly expressed and purified protein to test the effect of the various protein subunits on RNase P activity. Second, the archaeal system is simpler containing only four proteins instead of ten. Third, the archaeal proteins are smaller than their human homologs making them better for structural studies by NMR. Finally, since Methanothermobacter thermoautotrophicus and Pyrococcus furiosus are moderate and hyperthermophiles, respectively, their proteins should be thermostable and better behaved in solution. Given the homology between the human and archaeal proteins, structural/functional information gained into archaeal RNase P should also provide insight into human RNase P. Determining the three-dimensional structure of a protein is a first step towards gaining functional insights. In this work, I have determined the solution structure of the archaeal RNase P proteins Mth Rpp29 and Pfu Rpp21 using NMR techniques. Mth Rpp29 adopts an oligonucleotide/oligosaccharide binding (OB) fold while Pfu Rpp21 contains a zinc ribbon fold. Interactions between Mth Rpp29 and the P RNA subunit and between Pfu Rpp21 and Rpp29 provide a better understanding into the role of the individual proteins and information about the formation of the complex holoenzyme. The structural information gained through this study is a critical first step towards building a model of the holoenzyme, ultimately, yielding better insight into the evolution from an RNA-dominated enzyme in bacteria to a protein-dominated enzyme in archaea and eukaryotes.
Mark Foster (Advisor)
176 p.

Recommended Citations

Citations

  • Boomershine, W. P. (2005). Structure and interactions of archaeal RNase P proteins Mth Rpp29 and Pfu Rpp21 [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1117552357

    APA Style (7th edition)

  • Boomershine, William. Structure and interactions of archaeal RNase P proteins Mth Rpp29 and Pfu Rpp21. 2005. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1117552357.

    MLA Style (8th edition)

  • Boomershine, William. "Structure and interactions of archaeal RNase P proteins Mth Rpp29 and Pfu Rpp21." Doctoral dissertation, Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1117552357

    Chicago Manual of Style (17th edition)