Richard Bruce Hallick - US grants

The main objectives of the proposed research are (1) to determine the relationship between the rpoA, rpoB, rpoC DNA-dependent RNA polymerase subunit genes encoded in chloroplast DNA of Euglena gracilis and higher plants and the RNA polymerase activities of chloroplasts, (2) to define how the complex rpoB- rpoC transcription units are transcribed, and the resulting pre- mRNAs are processed and translated to yield polypeptide subunits, (3) to identify, isolate and characterize any additional chloroplast or nuclear localized genes for chloroplast RNA polymerases, and (4) to determine how the expression of the chloroplast RNA polymerase subunit genes is regulated during light induced organelle development. Emphasis will be placed on the importance of transcriptional vs post-transcriptional events in regulating the transcriptional vs post-transcriptional events in regulating the transcriptional status of the developig plastid. Chloroplast contain two types of transcriptional activity. In order to determine if the rpoA, B, and C genes encode subunits of either or both polymerase activities, polypeptide coding domains of the chloroplast rpoA, B, and C genes will being expressed in E. coli to yield fusion polypeptides for use in generating monospecific antibodies directed against the putatative RNA polymerase subunits. Antibodies will be used to immunopurify the enzymes, to identify subunits by Western blotting, and to specifically inhibit the different transcriptional reactions. Homogenous preparations of these enzymes should be obtained via immunoaffinity chromatography and FPLC, in addition to currently available purification procedures. Genes for RNA polymerase subunits that are not encoded by the rpoA, B, and C loci will also be studied. In order to determine how rpoC transcripts are processed, and if there are unusual features to this process such as alternat splice sites, cDNA clones of rpoC mRNA will be prepared and sequenced. Through the types of experiments described above and studies on the regulation of expression of the RNA polymerase subunit genes, and of the resulting enzyme activities, it will be possible to define many important fundamental aspects of RNA synthesis in cell organelles.

Many of the transfer RNA genes of chloroplast genomes have been found to be encoded in polycistronic operons. These operons can contain genes for other tRNAs, rRNAs, constitutively expressed proteins, or proteins whose expression is developmentally regulated. Regulation of chloroplast gene expression for most genes occurs posttranscriptionally. The regulatory mechanisms which could involve RNA processing events and/or translation initiation/elongation steps are not known. The focus of this proposal is to determine the RNA maturation pathways for developmentally regulated and constitutively expressed chloroplast polycistronic operons, and to determine the role of RNa processing steps, in any, in regulating organelle development. Some emphasis will be placed on mixed tRNA-mRNA operons, since precursor RNAs accumulate and tRNA processing must proceed mRNA translation. The main objectives of the proposed research are: (1) to determine the primary structure of precursor transcripts from polycistronic operons; (2) to determine if precursor transcripts and non- translated mRNAs that accumulate in proplastids are localized on polysomes or in ribonucleoprotein particles; (3) to characterize the RNa processing pathways for tRNA-mRNA mixed chloroplast operons containing constitutively expressed genes and photosynthetic membrane genes; (4) to determine if there are developmental specific RNA processing event during organelle biogenesis; (5) to purify selected RNA processing enzymes, and determine if the RNa developmentally regulated; (6) to characterize a novel class of chloroplast introns found only in constitutively expressed genes. In order to characterize organelle RNAs, we are developing a procedure for preparing cDNA clones of chloroplast transcripts. The procedure will be applicable to mature RNA species, primary transcripts, and RNa processing intermediates. The procedure will be general for transcripts from any source, not restricted to chloroplast RNAs, and will be applicable to polycistronic transcripts, including mixed tRNA-mRNA operons. These cDNAs will be subsequently expressed in vitro with T3 or T7 viral regulatory sequences and promoters as a means to generate substrates for in vitro characterization of RNa processing reactions.

The goal of the proposed research is to determine the biochemical mechanism of transfer RNA splicing in higher plant chloroplasts. Transfer RNA genes with 0.4-1.0 kbp introns, or intervening sequences, in their anticodon regions have been described for maize, tobacco, spinach and pea chloroplast genomes. Since these genes are expressed, the intervening sequence RNA must be spliced out of the primary transcript to produce functional tRNAs. The specific aims of this project are: (1) To obtain unspliced precursor transcripts of intron-containing chloroplast tRNA genes by intro transcription, and to characterize these precursor tRNAs by RNA fingerprinting methods. (2) To establish conditions for accurate splicing of these pre-tRNAs, and to characterize the requirements for intervening sequence excision and for exon and intron, and of the splice junction. (4) To partially purify the activity(ies) or factor(s) required for splicing in vitro and to determine their mechanism of action in splicing. Consequently, to determine whether different factors are required to splice different tRNA precursors (e.g., whether different endonucleases excise intervening sequences located at different positions in the anticodon loop). (5) To determine the secondary structures of these introns and to begin to investigate whether structures within the intron or the tRNA are required for excision or ligation. RNA splicing is a fundamental step in the expression of eukaryotic nuclear and organnellar genes. Chloroplast tRNAs, moreover, are an ideal model system for studies on organellar RNA splicing. The chloroplast tRNA introns may exhibit structural homology with a proposed class of fungal and plant mitochondrial mRNA introns. Each pre-tRNA, however, has only a single intron, and gives rise to a simplest system for biochemical investigation of the metabolism of a family of organellar introns. By comparing the mechanism of chloroplast tRNA splicing with those of other organisms, we hope to gain insight to the evolution of splicing mechanisms.

Many of the transfer RNA genes of chloroplast genomes have been found to be encoded in polycistronic operons. These operons can contain genes for other tRNAs, rRNAs, constitutively expressed proteins, or proteins whose expression is developmentally regulated. Regulation of chloroplast gene expression for most genes occurs posttranscriptionally. The regulatory mechanisms which could involve RNA processing events and/or translation initiation/elongation steps are not known. The focus of this proposal is to determine the RNA maturation pathways for developmentally regulated and constitutively expressed chloroplast polycistronic operons, and to determine the role of RNa processing steps, in any, in regulating organelle development. Some emphasis will be placed on mixed tRNA-mRNA operons, since precursor RNAs accumulate and tRNA processing must proceed mRNA translation. The main objectives of the proposed research are: (1) to determine the primary structure of precursor transcripts from polycistronic operons; (2) to determine if precursor transcripts and non- translated mRNAs that accumulate in proplastids are localized on polysomes or in ribonucleoprotein particles; (3) to characterize the RNa processing pathways for tRNA-mRNA mixed chloroplast operons containing constitutively expressed genes and photosynthetic membrane genes; (4) to determine if there are developmental specific RNA processing event during organelle biogenesis; (5) to purify selected RNA processing enzymes, and determine if the RNa developmentally regulated; (6) to characterize a novel class of chloroplast introns found only in constitutively expressed genes. In order to characterize organelle RNAs, we are developing a procedure for preparing cDNA clones of chloroplast transcripts. The procedure will be applicable to mature RNA species, primary transcripts, and RNa processing intermediates. The procedure will be general for transcripts from any source, not restricted to chloroplast RNAs, and will be applicable to polycistronic transcripts, including mixed tRNA-mRNA operons. These cDNAs will be subsequently expressed in vitro with T3 or T7 viral regulatory sequences and promoters as a means to generate substrates for in vitro characterization of RNa processing reactions.

This project is a study of the structure, function, and splicing mechanisms of the group II and group III introns and twintrons ("introns-within-introns") of the plastid genomes of Euglena gracilis and related protists. These organisms are the richest known source of group II introns, and have the only known group II and group III twintrons. The specific aims include: (1) Characterization of novel group II and group III introns and twintrons in species related to Euglena gracilis as a means to explore (i) possible "founder" events of intron invasion, (ii) whether group II and group III intorns shared a common conserved structural motifs required for RNA splicing; (2) An investigation of the role of the group III intron spicing, and (3) Analysis of cis-acting domains of group II and group III intorns required for in vivo splicing in transgenic I. gracilis chloroplasts. This aim will be expanded to include site-directed mutagenesis of intron maturases if sufficient technical progress in plastid transformation is achieved. This work is addressed at understanding fundamental questions about the origins and evolution of introns and twintrons. A working hypothesis is that group II and group III introns, as well as nuclear pre-mRNA spliceosomal intorns, may have all shared a common evolutionary ancestor. Therefore, studies on unusual variants among the smallest group II and group III introns should be relevant to understanding the RNA structures at the heart of all splicing reactions.