 |
Margaret J. HollingsworthMolecular Biology: Post-transcriptional regulation of gene expression Associate Professor PhD 1983 University of
Colorado/Boulder |
|
Margaret J. HollingsworthMolecular Biology: Post-transcriptional regulation of gene expression Associate Professor PhD 1983 University of
Colorado/Boulder |
Margaret J. Hollingsworth
Department of Biological Sciences
653 Hochstetter Hall
State University of New York at Buffalo
Buffalo, NY 14260
(716) 645-2363 ext: 196
To send e-mail: hollings@buffalo.edu
Research in our lab concentrates on posttranscriptional processes that affect gene expression in the chloroplast. Our research has its primary focus on cis and trans-acting factors involved in posttranscriptional control of chloroplast gene expression.
Publications from 1982 on can be found at this link
RNA-PROTEIN COMPLEXES AND CHLOROPLAST GENE EXPRESSION
. . . . . . In all living systems, any process involving RNA is affected by the formation of RNA-protein complexes. Complexes that form in untranslated regions (UTRs) of mRNAs are especially common in chloroplasts, where they mediate several mechanisms that control post-transcriptional aspects of gene expression. The focus of our research is the study of 5'UTR-protein complexes in land plant chloroplasts. Our research on chloroplast 5'UTR-protein complexes has three themes: cis-acting elements, trans-acting factors, and function.
cis-acting elements To simplify analysis of 5'UTR-protein complexes, we use a single representative chloroplast 5'UTR as the baseline in all our experiments. It is derived from the atpI gene, which encodes the CFo-IV subunit of the ATP synthase complex and is the second gene in the chloroplast large ATP synthase gene cluster. Affinity of the binding proteins and the stability of the complex formed with atpI 5'UTR are similar to that for many other chloroplast 5'UTRs. Thus insights into complex formation discovered with the atpI 5'UTR will be applicable to those formed with 5'UTRs from many other chloroplast genes. A phylogenetic comparison of this UTR among angiosperms revealed that there are two especially highly conserved regions of sequence, which we have termed Con1 and Con2 (see figure). In tobacco, Con1 and Con2 match 81% and 85% (respectively) of the consensus sequence generated from all angiosperms whose sequence is currently available.
. . . . . . The atpI 5'UTR can be separated into two binding elements, each containing one of the two phylogenetically conserved sequences. Competition binding analysis has revealed that different proteins bind the two elements. Current research on cis-elements concentrates on identification of secondary/tertiary structures that affect complex formation in vitro.
trans-acting factors Competition binding assays were used to determine whether the proteins that bound the atpI 5'UTR were specific only for that mRNA. Results from those experiments showed that the same chloroplast proteins bind 5'UTRs from mRNAs encoding a diverse assortment of chloroplast polypeptides. Thus we hypothesize that these 5'UTR-protein complexes may serve a global function in regulation of chloroplast gene expression. Current research on 5'UTR binding proteins entails isolating them and determining their individual effects on chloroplast gene expression in vivo.
Function Chloroplast transformants are used to investigate 5'UTR function in vivo. Wild-type and variants of the atpI 5'UTR are placed upstream of a reporter gene and transformed into tobacco chloroplast genomes. Transgenic plants are analyzed for translation and abundance of the reporter RNA. Preliminary data show that the 5'UTR strongly affects translation within the chloroplast. There appear to be some 5'UTR-mediated affects on RNA abundance as well. Current research involves transformation studies with further variations in the 5'UTR, to better define specific 5'UTR sequences and structures involved in post-transcriptional regulation of chloroplast gene expression.
Land plant 5'UTRs have conserved
sequences and (predicted) structures
This structure is a computer-derived consensus between the tobacco and Arabidopsis atpI 5'UTRs.
(Mathews et al, www.rna.chem.rochester.edu )