Type of Document	Thesis
Author	Cavnar, Katie M.
Author's Email Address	eggers@bio.fsu.edu
URN	etd-08192004-152949
Title	Development Of A DNA Probe Assay For Rare Transfer Rnas And Its Use In Measuring The Expression Of The Argx Gene In Escherchia Coli
Degree	Master of Science
Department	Biological Science, Department of
Advisory Committee	Advisor Name Title Lloyd Epstein Committee Member Robert Reeves Committee Member Thomas Keller Committee Member
Keywords	Rare tRNAs Sandwich Hybridization Assay ArgX Gene
Date of Defense	2004-07-20
Availability	unrestricted
Abstract In Escherichia coli, the argX gene is the first gene in a cluster with three other tRNA genes. The tRNA products of the three downstream genes, hisR, leuT and proM are abundant in E. coli, and all four of the tRNAs are processed from the same precursor RNA transcript. The argX promotor appears to be a strong promotor and is preceded by an Up element. By all accounts, the gene product of argX, tRNAarg3 should be a major tRNA. Like the three downstream genes in this cluster, argX must be transcribed at a high level because of its location at the beginning of the gene cluster. However, tRNAarg3 is considered a rare tRNA in E. coli. Its low level is correlated with the low codon usage of its corresponding codon, CGG. It appears that the nucleotide sequence just upstream of argX can base pair with the 5’- end of the tRNA. This alternative secondary structure in the precursor RNA transcript may prevent the maturation of the tRNAarg3 by disrupting the base pairing of the aminoacyl-tRNA stem. This structure is required by ribonuclease P for maturation of the 5'-ends of all tRNAs and also prevents the 3'-exonucleases from digesting into the tRNA from the CCA (3'-) end. Different constructs of the argX gene have been cloned into high copy number plasmids. These constructs differ in the nucleotides just upstream of the argX gene. Deletion of nucleotides results in higher expression of tRNAarg3 and the loss of an alternative secondary structure involving the 5’–end of the tRNA. The data suggests alternative secondary structure formation during transcription may in part be responsible for tRNAarg3 abundance in E. coli. A tRNA assay has been developed to measure the levels of rare tRNAs from extracts. This assay is both sensitive and specific and demonstrates its ability to detect low levels of tRNAs.
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