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|Title:||Investigation of Regulation of Transfer RNA Gene Expression in Mammalian Cells: Utilization of a Human Nonsense Suppressor Transfer RNA|
|Authors:||Tapping, Ian Richard|
|Advisor:||Capone, John P.|
|Abstract:||<p>The regulation of eukaryotic transfer RNA gene expression serves largely to adapt the abundance of each isoacceptor tRNA species to the codon frequencies and amino acid usages particular to different cell types. tRNA genes are independently transcribed and, even within an isoacceptor family, can display considerable differences in transcriptional activity in vitro which cannot be ascribed to the highly conserved nature of their intragenic promoter elements. In this regard, it is well documented that expression of transfer RNA genes can be dramatically influenced in a wide variety of species by 5' flanking sequences. Unfortunately, it remains uncertain what role the majority of these modulatory elements play in their normal cellular context since most of these studies have utilized in vitro transcription systems whose ability to faithfully reproduce cellular conditions is questionable. Unfortunately, it is difficult to monitor the expression of an individual tRNA gene in vivo since the gene for any particular isoacceptor is reiteratedin the eulcaryotic genome. To overcome this, a nonsense suppressor transfer RNA gene has been utilized to study the regulation of tRNA gene expression in mammalian cells. Specifically, the functional expression of a human serine amber suppressor tRNA gene has been quantified in vivo by assaying its ability to suppress an amber nonsense mutation in the Escherichia coli chloramphenicol acetyltransferase gene following cotransfection in mammalian cells.</p> <p>Through utilization of the aforementioned in vivo assay the suppression activity of a series of upstream deletion and substitution mutants of the human serine tRNA gene was determined. Mutant genes in which the 18 nucleotides 5' proximal to the coding region were deleted and replaced with heterologous sequences, were 2 to 5 fold more active in vivo in comparison to the wild type gene. The serine tRNA gene constructs were also transcribed in vitro using HeLa cell nuclear extracts. The strong correlation between the transcriptional activity in vitro and functional expression in vivo of the various mutants indicates that this negative element acts by modulating the transcriptional activity of the serine tRNA gene and suggests that this element plays a physiologically relevant role within the mammalian cell. Second template competition experiments demonstrate that the element reduces the ability of the serine tRNA gene to stably sequester limiting transcription components. The results from several insertion mutants, which effectively alter the orientation and upstream position of this negative element, suggest that it acts in a dominant negative manner in vivo and in vitro.</p> <p>The precise mechanism by which extragenic flanking sequences modulate tRNA gene transcription is unknown and a direct comparison of identified modulatory regions has failed to reveal conserved sequence elements that could be ascribed to being positive or negative transcriptional modulators. One possibility is that specific regulatory factors bind these upstream sequences and attenuate tRNA gene transcription, but the isolation of any such activity has been difficult to demonstrate since the composition of higher eukaryotic tRNA gene transcription complexes is unknown. As an alternative approach to examining this possibility, an oligonucleotide containing the recognition site for the Escherichia coli lac repressor was inserted at various positions in the 5' flanking region of the human serine tRNA gene and the consequences of binding lac repressor on in vitro transcription by RNA polymerase III was investigated. lac repressor prebound to operator sites centered at positions -9, -15, -35, and -37 upstream of the mature tRNA coding region completely inhibited transcription by interfering with the formation or stability of transcription complexes. lac repressor also inhibited transcription of serine tRNA gene derivates containing operator sites at -9 and -15 when added following transcription complex assembly or during ongoing synthesis, but had no effects on the other tRNA gene derivatives if added subsequent to complex assembly. lac repressor prebound at position -43 and -46 partially inhibited transcription and redirected initiation to sites farther downstream. These results show that the functional human RNA polymerase III transcription complex extends at least 35 nucleotides upstream of the tRNA gene coding region and that the sequences surrounding the transcription start site remain accessible to DNA-binding proteins throughout multiple rounds of transcription. Normal transcription was restored with the addition of the allosteric inducer IPTG demonstrating that these effects require the continued presence of bound repressor protein. In addition, lac repressor inhibited the functional expression of the human serine tRNA gene in vivo since inclusion of a plasmid expressing the lac repressor in the above cotransfection assay resulted in inhibition of suppression activity of lac operator-linked genes. This effect was also alleviated with IPTG. These results demonstrate that a heterologous DNA binding protein bound to 5' flanking sequences can be used to regulate the expression of a mammalain tRNA gene in vivo and in vitro. Although artificial in its approach, this study shows that this is a potential mechanism by which naturally occcuring regulatory proteins may modulate the expression of cellular tRNA genes. In addition, these experiments revealed information regarding the upstream spatial arrangement and topological boundaries of functional mammalian tRNA gene transcription complexes.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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