Studies on methlation and primary structure of the transfer RNA of mycobacterium

Abstract

Transfer RNA (tRNA) molecules are perhaps the smallest polynucleotides having profound biological functions within the cell. They serve as suitable systems to understand protein–nucleic acid interactions owing to the variety of interactions they participate in, with several enzymes and protein factors of the protein synthesizing machinery, including ribosomal proteins. Detailed studies have revealed a paradoxical structural requirement for tRNAs. They must retain a significant degree of sequence and structural conservation, since they participate in interactions common to all tRNAs, such as ribosome binding. On the other hand, they must possess sufficient uniqueness to maintain translational fidelity and perform additional regulatory functions. With these constraints, some researchers have suggested that sequence divergence and post transcriptional modifications of tRNA follow an evolutionary pattern. Our laboratory has been involved in studying various aspects of the biochemical processes of bacteria of the genus Mycobacteria. The present project involves a molecular level study of tRNA structure and metabolism. Mycobacteria is an interesting genus with a disputed taxonomic position, classified under the family Mycobacteriaceae and the order Actinomycetales. The present work includes analysis of the base modifications in the tRNA of Mycobacterium smegmatis (a saprophytic species of Mycobacteria), with special emphasis on methylation and sequencing of the initiator tRNA to determine the exact positions of modified nucleosides and its primary structure. The first section of the thesis (Chapter I) provides a survey of literature pertaining to relevant aspects of tRNA research.

Minor Base Composition Analysis (Chapter II) The elucidation of minor base composition in M. smegmatis tRNA was taken up first. In these studies, in vivo labelled (^32P or ^14C) total tRNA was subjected to enzymatic digestion (with RNase) or chemical digestion (0.3N KOH). The resulting mononucleotides were analysed using:

Two dimensional thin layer chromatography, High voltage paper electrophoresis, and High performance liquid chromatography (HPLC).

The major observation was that ribothymidine (5 methyl uridine) is absent in M. smegmatis tRNA, whereas it contains 1 methyladenine, which is uncommon among prokaryotes. RNase T and alkali digests were examined for sugar methylated nucleosides, but none could be detected using this method. HPLC comparison of tRNA from various Mycobacteria-M. smegmatis, M. lacticola, M. phlei-and from M. smegmatis infected with phage I revealed the presence of 1 methyladenine and 7 methylguanine and the absence of ribothymidine in all species.

Detection of Modified Nucleotides (Chapter III) A new method was developed for identifying modified nucleotides in total tRNA samples. This involved:

Limited formamide digestion of tRNA at 98°C, 5 ^32P post labelling of resulting oligonucleotides using T polynucleotide kinase, Separation by sequencing gels following the method of Kuchino et al. (1979).

This highly sensitive procedure allowed assignment of modified bases to specific regions of the tRNA cloverleaf. The following modifications were detected:

1 methylguanine (m¹G) 2 O methylcytosine (Cm) 2 O methylguanine (Gm) 2 O methylpseudouridine ( m)

Some modified nucleotides remained unidentified. 1 methyladenine (m¹A) occurred in more than one position in certain tRNAs.

Sequencing of M. smegmatis Initiator tRNA (Chapter IV) To determine the disposition of modified bases and the sequence itself, complete sequencing of the initiator tRNA was performed. Initiator tRNA was chosen because these molecules possess hallmark features that distinguish prokaryotic and eukaryotic systems. Purification was achieved using:

Reverse phase chromatography Polyacrylamide gel electrophoresis

Sequencing methods used included:

Hot formamide digestion (Kuchino method), Donis Keller enzymatic digestion, Peattie’s chemical sequencing method.

Key features of M. smegmatis initiator tRNA include:

Presence of U CG sequence in loop IV (instead of U CA in prokaryotes or AUCG in eukaryotes), Presence of a C–U pair at the 5 terminus.

Modified nucleoside positions determined include dihydrouridine at 17, 7 methylguanine at 46, pseudouridine at 55, and 1 methyladenine at 58.

1 Methyladenine tRNA Methyltransferase (Chapter V) Since m¹A was found in multiple locations, the enzyme responsible was investigated. An in vitro methylation assay was standardized using:

E. coli (w) tRNA (lacking m¹A), ^14C SAM as methyl donor.

M. smegmatis extracts methylated only m¹A in E. coli tRNA, while E. coli extracts formed ribothymidine in M. smegmatis tRNA. Two distinct peaks of m¹A forming methyltransferase activity were detected on ion exchange columns. The major fraction was purified to homogeneity by AMP Sepharose affinity chromatography. Electrophoretic and immunological methods confirmed purity.

Inhibition Studies (Chapter VI) Ethionine and SIBA inhibited both growth and methylation by over 95%. Aminoacylation studies showed enhanced charging efficiency with in vitro methylated tRNA. Melting profiles suggested that methylation destabilizes tertiary structure at lower temperatures.

Summary of Findings

M. smegmatis tRNA lacks ribothymidine and contains unique modifications such as m¹A. Modified nucleosides were assigned to specific structural regions. Initiator tRNA displays a mixture of prokaryotic and eukaryotic features. Two distinct m¹A methyltransferases may recognize different tRNA sequence contexts. M. smegmatis tRNA exhibits traits characteristic of both bacteria and higher organisms.