Marvin R. Paule - US grants

This is a proposal to study the mechanism of rRNA transcription and its regulation in eukaryotes. An in vitro transcription system made up of cloned rDNA, homogeneous RNA polymerase I (RNAP I) and an auxilliary protein (transcription initiation factor(s)-TIF) is being utilized: (1) to determine the number of TIFs, and to purify them to homogeneity, (2) to determine in greater detail the role played by the TIF in initiation of transcription, (3) to determine how the TIF and RNAP I interact with each other and with the template and (4) to describe kinetically the events involved in the assembly of the initiation complex. DNA footprinting and chemical modification-protection experiments will be used to determine protein-DNA contacts. Chemical cross-linking experiments will be used to determine protein-protein interactions. Kinetic measurements will be used to determine the kinetic mechanism of initiation. Monoclonal antibodies to the RNAP I subunits and the TIF(s) will be produced to aid in the analysis. Transcription of rRNA gene expression is developmentally regulated in Acanthamoeba by modification of RNAP I. Experiments to identify the chemical nature of the modification and the enzyme catalyzing it are proposed. Exactly which step in the process of initiation is affected by the modification is also to be studied utilizing the techniques proposed to study the mechanism of initiation.

The major objective of this proposal is the isolation and characterization of ribosomal DNA from Acanthamoeba castellanii. Recombinant DNA techniques will be used to isolate the gene, restriction endonuclease mapping and R-loop mapping will be used to determine the topology of the isolated DNA fragments. DNA sequencing of promotor and terminator regions will be carried out and compared to similar sequences determined in other eukaryotes.

In eukaryotic cells, the promoter for the ribosomal RNA gene consists of several regions: a core and a varying number of upstream elements. The role of the core is to bind one or more trans-acting transcription factors. In Acanthamoeba, a single factor, TIF, binds to a defined sequence in the core. In this laboratory, TIF has been very highly purified, and its interaction with the DNA has been characterized in detail by mutagenesis of the promoter and footprinting techniques. Interactions between TIF and polymerase also have been characterized by the same methods. Among the significant findings are that TIF directs RNA polymerase I to bind over the transcription start site by protein-protein contact; polymerase makes no sequence-specific contacts with the DNA during binding. Further, rRNA transcription is regulated by modification of the RNA polymerase, and the modification prevents polymerase from binding to the promoter suggesting a possible effect on the TIF-polymerase interaction. In order to more fully understand the mechanism of initiation and regulation of rRNA expression, the cloning of the TIF gene is proposed. The purified TIF will be partially sequenced and an oligonucleotide probe synthesized from the predicted DNA sequence. A genomic library, which because of the small genomic complexity of Acanthamoeba consists of only 9200 plaques, will be screened for the TIF gene. Following sequencing, the genomic clone will be used to screen a cDNA library. The cloned TIF gene will be mutagenized, transcribed and translated in vitro, and the functional domains of the protein identified using several assays. Template commitment, footprinting, and gel shift assays will be used to evaluate DNA binding. Transcription run-off assays and DNA footprinting will be used to identify TIF-mediated polymerase binding and activation. Footprinting assays will determine the effects of mutant TIF binding on DNA conformational changes associated with the wild-type factor binding. In the long term, the details of the interaction of TIF and polymerase will be evaluated using site-specific point mutagenesis of the TIF gene and cloned RNA polymerase subunit gene(s).

Determination of the sequence of amino acids is the first step toward the study of cloning and structure-function relationships of proteins. DNA of known base sequence can be synthesized if the amino acid sequence of a protein is known. Genes for several proteins are proposed to be cloned in the Biochemistry Department; these include actin depolymerization factor and an rRNA transcription initiation factor. Amino acid sequence is also essential to find the site of proteins damaged by oxyradicals and hydrogen peroxide; for the region of neurotoxin-acetylcholine receptor binding; to elucidate the active center of snake hemorrhagic toxins; and to determine the regulatory site of phosphorylation in actin depolymerizing factor. The active sites of the RNA polymerases from E. coli and bacteriophage T7 are also proposed to be studied. Unfortunately, there is no protein sequencer at Colorado State University. At the moment we are relying on a facility at a neighboring state university. Our ability to do protein research is impaired by the lack of more direct and timely access to this instrumentation. Acquisition of a sequenator will enhance the efficiency and quality of protein research at Colorado State University.

5S RNA and ribosomal RNA transcription rates, and expression of ribosomal proteins are closely tied to cellular growth rate in order to balance elaboration of ribosomes. This proposal is aimed at studying the mechanism of coordinating expression from these genes, which are transcribed by distinct RNA polymerases. In Acanthamoeba, rRNA transcription is regulated by polymerase I modification, and recent results show that 5S RNA transcriptional shutdown is accompanied by a loss in TFIIlA transcriptional and DNA binding activity in nuclei. The mechanism of this loss is unknown. This study will examine the change in TFIIlA activity by several approaches: The level of TFIIlA protein present in the whole cell will be revealed by Western blot analysis. A change would indicate that there is less TFIIlA in the cell. If so, an investigation of the mechanism for this change will involve cloning the gene for TFIIlA and determining whether TFIIlA mRNA declines in parallel with TFIIlA protein. If so, nuclear run off experiments will determine whether the TFIIlA mRNA level is regulated transcriptionally. If TFIIlA is regulated transcriptionally, an investigation into the mechanism of this regulation will be carried out. First, the promoter of the cloned TFIIlA gene will be dissected using in vitro transcription. If TFIIlA mRNA levels are not regulated transcriptionally, TFIIlA mRNA association with polysomes will be examined to determine if translation is specifically hindered. Alternatively, if the overall cellular level of TFIIlA remains constant, the cellular localization of TFIIlA, eg. as a complex with 5S RNA in the cytoplasm, will be investigated by analyzing the distribution of TFIIlA in nuclei and the cytoplasm, and by determining the amount of 7S RNP and 42S RNP in the cytoplasm at different stages of cellular development and 5S RNA transcriptional activity. If the amount of TFIIIA protein present in nuclei is constant, but only the DNA binding activity of the factor is altered, then TFIIIA must be modified so that it cannot interact with the 5S RNA gene. An investigation of modification will be carried out by structural comparison of TFIIlA from active and inactive cells. Later in Acanthamoeba development, the activity level of TFIIIC is reduced, presumably to shut down other type 2 polymerase III transcription. We will investigate the mechanism of this change using an approach similar to the above study of TFIIIA.

DESCRIPTION: This is a proposal to continue studies of eukaryotic ribosomal RNA transcription initiation and regulation. RNA polymerase I (pol I) transcribes this gene. The rRNA gene of the eukaryotic organism being studied, Acanthamoeba, has properties making it especially good for the biochemical studies proposed; properties no other pol I system currently exhibits. In particular, the unusual stability of the complex between its core promoter and transcription factor allow analysis by techniques not available in other systems. Recent findings lead logically into this proposal. The entire rRNA intergenic spacer was sequenced, pol I enhancers were identified, and shown to bind a vertebrate UBF homolog. The TATA-binding protein (TBP)- containing initiation factor (TIF-IB) was purified to homogeneity, its subunits (TAFIS) identified, and TBP's distinctive role in pol I transcription revealed. NTP beta-gamma hydrolysis was shown not to be needed for initiation, and the DNA melting process by pol I was examined in detail. Site-direct photocross-linking of TIF-IB and pol I to promoter DNA resulted in mapping the topology of TAFIS and TBP on the rRNA promoter. Coupled with STEM pictures, this study has given insights into the activated promoter complex never before detailed, and gave significant insights into how pol I promoters function. Regulatory studies implicated the pol alpha subunit homolog (AC39) in regulation, and inroads into its cloning and sequencing were made. Funds are now requested to attain the following aims: (A) Protein-protein and protein- DNA interaction will further detailed using new sophisticated photoaffinity probes, heterobifunctionals cross-linking agents, and high resolution STEM. (B) The role, if any, of several potential additional transcription factors which influence rRNA transcription will be evaluated. These include a Ku-like factor and a factor required for stable complex formation on the core promoter. (C) The regulatory mechanism of rRNA transcription will be investigated, placing emphasis on the chemical events resulting in the inactivity of pol I purified from transcriptionally inactive cells. Investigation of the known modification of the AC39 subunits, and its role in regulation, will be a particular aim. (D) Genes for the TAFIS and key pol I subunits will be cloned. (E) A method for transient and stable transformation of Acanthamoeba will be developed.

DESCRIPTION (from the application): This laboratory has been a major contributor to our understanding of the mechanism of rRNA transcription initiation and regulation. However, the experimental system we have studied does not exhibit some characteristics of higher eukaryotic rRNA transcription, in particular, it does not require an upstream activator or upstream promoter elements. Because S. cerevisiae does not require these, and further has very facile and powerful genetics, a biochemical and genetic study of yeast is proposed. Over the past decade, the genetics of rRNA transcription has been effectively exploited in yeast, with isolation of genes defining three transcription factors, UAF, CF, and Rrn3p, which are needed in addition to polymerase I and TATA-binding factor for activated transcription. The biochemistry of the system has lagged significantly behind the genetics, leaving a number of mechanistic questions unanswered. These will be investigated. The structure of the committed and preinitiation complexes will be revealed by a battery of approaches, including five footprinting methods and site-specific DNA-protein photo-cross-linking, drug inhibition studies, DNA topological analyses and genetic screens for TBP mutants. These studies will determine the architecture of the preinitiation complexes, determine the groove of the DNA primarily contacted by proteins, and the path of the DNA in these complexes. Yeast two-hybrid analyses will reveal the protein-protein interactions in the complexes. The mechanistic role of TBP in activated transcription will be elucidated, with emphasis on its mechanism of interaction with DNA, and resulting DNA topology changes. In addition, the functional mechanism of several of these factors will be investigated. In particular, the mechanism of the transcription factor implicated in the regulation of rRNA transcription, Rrn3p, will be determined; assays to determine if it mediates polymerase recruitment, DNA melting or promoter clearance will be used. The RNA polymerase I and the transcription factor subunits that interact with each other to mediate pol I recruitment will be determined both biochemically and genetically. The potential role of polymerase subunit or Rrn3p modification in regulation will be investigated. These studies will yield the most complete description of the biochemistry and genetics of any eukaryotic rRNA transcription system.

The Molecular Biosciences Summer REU Site at Colorado State University is a 10-week program that exposes undergraduates to the broad range of research areas and approaches that constitute the modern discipline of biochemistry and molecular biology. The program begins the Tuesday after Memorial Day and ends in the first week of August. Twelve students will be selected to work with mentors from five departments. Students are involved in their own research project where they learn how to formulate and test hypotheses, design and execute experiments, analyze results in the context of the hypothesis, and use state-of-the-art techniques and instrumentation. Interactions with faculty, graduate students, postdoctoral fellows, and professional research staff provide a mechanism for learning about the latest advances in biochemistry and molecular biology. Students receive a stipend, free housing in a university residence hall or apartment building, a meal allowance, and an allowance to defray travel to/from the site. At the end of the program, students present their research results orally and through a poster, and produce a final report in manuscript form. In addition to research, students are engaged in social activities such as white water rafting and hiking in Rocky Mountain National Park. Field trips to nearby government labs and biotech companies as well as workshops on GRE preparation and scientific careers are also planned. Students with limited opportunities for research and those from groups under-represented in science are especially encouraged to apply. For further information, students may visit http://www.bmb.colostate.edu/reu.cfm, or contact Marti Stokes at 970-491-5602 or Marti.stokes@colostate.edu or reubiochem@colostate.edu or Paul Laybourn at 970-491-5100 or Paul.Laybourn@colostate.edu.