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GENERAL  ARTICLE
Modified Bases in Mycobacterial Transfer RNA
Vani Brahmachari
Vani Brahmachari did her
doctoral research at the
Microbiology and Cell
Biology Department, IISc,
under the guidance of
Prof. T Ramakrishnan.
She is presently a
professor at Dr. Ambedkar
Centre for Biomedical
Research, University of
Delhi. Her research focus
is in the area of epigenetic
mechanisms and developmental biology. She is also
engaged in mining the
human genome to discover
genes involved in epigenetic regulation and on
functional analysis of M.
tuberculosis genome.
The presence of post-transcriptionally modified bases in
nucleic acids is associated with various regulatory mechanisms. The transfer RNA is particularly rich in modified
bases. The article discusses some of Prof. T Ramakrishnan’s
contributions to the filed in the context of the times, which
illustrate his foresight.
There are more than 100 modifications reported for transfer RNA
(tRNA) which entered the field of molecular biology when Francis
Crick proposed the role for an adaptor molecule to decode the
information contained in DNA into proteins. Nature has invested
considerably in generating and maintaining modified bases including all the genes coding for catalytic functions to modify
nucleotides after transcription. It was the belief that such a
strategy indeed would have an important role in the biology of
Mycobacterium that Prof. Ramakrishnan took up research on
modified bases in RNA in the bacterium, anticipating that the
modifying enzymes could be potential drug targets. I will try and
summarise his work which led to the sequencing of the initiator
tRNAfMet, and the elucidation of unique features of Mycobacterial tRNAs.
Transfer RNA – The Adaptor Molecule
Keywords
Mycobacterium tuberculosis,
tRNA, modified bases.
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Transfer RNAs are ubiquitous and are considered ancient molecules that directly interface between the information contained
in DNA and the amino acid sequence of a protein (Figure 1).
They are small in size, typically between 73 to 93 bases, most
often 76 nucleotides, approximately 25kd in molecular mass.
They were discovered as soluble ribonucleic acid intermediates
in protein synthesis by Paul Zamecnik in 1958. Alanine tRNA of
yeast was the first to be sequenced by Holley and coworkers.
Based on the sequence, they proposed the famous cloverleaf
structure of tRNA by applying the criteria of maximum base
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GENERAL  ARTICLE
(A)
(B)
Figure 1. Transfer RNA molecules interface information transfer between DNA and protein.
(A) Charging of cognate amino acid to specific tRNA by aminoacyl-tRNA synthetase. Energy
released by ATP hydrolysis is utilized, forming AMP and pyrophosphate (PPi).
(B) The amino acid is carried at the 3’CCA end of tRNA, attached to the 3’OH of the ribose moiety
and placed on the ribosome, through recognition of the codon on mRNA. P and A stand for entry
sites (P site and A site) for aminoacyl-tRNA on ribosomes. Only the initiator tRNA directly enters
the P site on ribosomes while all other aminoacyl-tRNA enter A site.
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GENERAL  ARTICLE
Figure 2. Cloverleaf structure of initiator tRNA of Mycobacterium smegmatis.
This also illustrates the
general structural features
of transfer RNA described
in the text. The features
unique to initiator tRNA of
Mycobacterium are marked: the presence of 1-methyl adenine (m 1 A) and
uracil in place of thymine
found in almost all other
tRNAs.
pairing. The cloverleaf structure of tRNA elegantly fits an adapter molecule with attachment of
specific amino acid on its 3’end and the anticodon presented as a single-stranded loop that can
interact with the codon on mRNA. The L-shaped three-dimensional structure of tRNA was
deciphered by high resolution X-ray crystallography by Alexander Rich and coworkers. The
cloverleaf structure has four stems and three loops; the stems being designated either by the
modified base they carry or their function (Figure 2). A highly conserved single-stranded
sequence N73CCA-OH (N for any of the four nucleotides), is present at the end of the acceptor
stem-loop and the terminal adenosine contains free 2’ and 3’ hydroxyl groups where an amino
acid is attached. Two or three base pairs in the acceptor stem vary between tRNAs and are
referred to as the 'discriminator’ nucleotide. The first base from the 5’end and the 72nd base
remain unpaired in prokaryotes while they are always paired in eukaryotes. Then there is the
TC stem-loop with psudouridine (, Figure 1B) and ribothymidine. The anticodon stem-loop
contains the three-lettered anticodon as N35N36N37 that directs the interaction of tRNA with
the codon on the mRNA. The D stem-loop contains dihydrouridine. The L-shaped threedimensional structure comes about by stacking acceptor stem-loop on TC stem-loop and D
stem-loop on anticodon stem-loop.
Transfer RNAs are coded in the bacterial genome often as operons with a common promoter and
transcribed as a long transcript from which each tRNA is cleaved by RNAse activity. For
example, tRNAArginine, tRNAHistidine, tRNALeucineand tRNAProline exist as an operon in E. coli. The
role of tRNA in protein synthesis is very critical to maintain the accuracy of information transfer
from the DNA to protein. The process of attachment of an amino acid to the right tRNA is
catalysed by aminoacyl tRNA synthetases (aa-tRNA synthetase) in two steps. There are 20
different enzymes to specifically charge the amino acid to the right tRNA with matching
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anticodon. In the first step the amino acid is activated by ATP to form tRNA synthetase (amino
acid-AMP) which in the second step is loaded on tRNA to form aminoacyl tRNA (aa-tRNA).
One may wonder as to how this specificity is maintained. Nobel Laureate Chrisitan de Devu
commented that the second genetic code is written into the structure of aminoacyl-tRNA
synthetase [1]. He also adds “….(this) code is probably nondegenerate and could be older and
more deterministic than the genetic code”. While the codon–anticodon interaction is a single
language of nucleic acids, the specificity of charging the right amino acid on the cognate tRNA
is 'bilingual', involving a protein and a nucleic acid. The major role played by aa-tRNA sythetase
in maintaining the fidelity in charging of tRNA with the right amino acid was subsequently
deciphered. It is accepted that the nature of the base at 73 position and one or two base pairs
among the first four in acceptor stem are important for fidelity. It was also shown that anticodon
has no role in achieving this specificity, as specific amino acylation can take place even when
the anticodon stem-loop is removed.
Modified Bases in Transfer RNA
The presence of modified bases in tRNA was suspected when amino acid accepting activity of
E. coli tRNA was found to be resistant to ribonuclease, an enzyme that can hydrolyse RNA.
This work was carried out by Susumu Nishimura who was earlier at the National Cancer Centre
Research Institute, Tokyo and presently at the University of Tsukuba, Japan. Subsequently, in
collaboration with Har Gobind Khorana’s group, by sequencing E. coli tRNATyrosine he found
that tRNAs have unusual bases. The presence of multiple codons for one amino acid demands
a calculated mispairing of bases referred to as ‘wobble base pairing’, which is facilitated by
modified nucleotides at the first position of the anticodon. There are more than 100 modifications reported for tRNA from different sources (Figure 3). Nishimura and his group carried out
structural analysis of several modified nucleotides. After a sabbatical at Japan in 1978, Prof.
Ramakrishnan (TR as he was fondly addressed by his students) established collaboration with
Nishimura and analysed modified bases in tRNA and ribosomal RNA in Mycobacteria.
TR had a project which was directed at analyzing whether Mycobacterium tuberculosis as well
as other non-pathogenic species of Mycobacteria have any base modifications. The goal was to
explore if the uniqueness of base modification, if any, in Mycobacteria, which could be
exploited for chemotherapy of tuberculosis. The work began with an observation that enzyme
fractions from M. smegmatis, could methylate total tRNA from E. coli. The results were
reported in a paper presented by M S Shaila and TR at a symposium organized by the
Department of Atomic Energy at Mumbai. Though the substrate tRNA in these reactions was
completely modified within E. coli cells, enzymes from M. smegmatis could add methyl groups
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Figure 3. Structure of modified bases found in tRNA. Representative modifications of each of the
four bases are shown. Lysidine and Q base are complex modifications of cytidine and purine
respectively. Note that both base and sugar modifications are known to occur in tRNA. The
structures are taken from http://library.med.utah.edu/RNAmods available in the public domain.
to it. This suggested that the modified base composition of tRNA in M. smegmatis is different
from that of E. coli tRNA. Similarly in a reciprocal reaction, the enzymes from E. coli also
found the tRNA from M.smegmatis lacking some modifications. This led to the discovery that
M. smegmatis tRNA contains 1-methyl adenine (m1A) (which is absent in E. coli tRNA) but
lacks ribothymidine in the TC loop. A detailed analysis of base composition of total tRNA
from M. smegmatis was taken up [2]. E. coli tRNAs are enriched in modified nucleotides, while
they are considerably less in Mycobacterial tRNAs.
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It is well known that thymine is almost exclusively found in DNA; RNA molecules contain
uracil in its place. However as seen in the cloverleaf structure of tRNA (Figure 2), there is
thymine residue in almost all tRNAs in the TC loop. The difference between uracil and
thymine is the presence of –CH3 group on the 5th carbon in the pyrimidine ring in the latter.
What is interesting is that this residue is not incorporated into tRNA during transcription of
tRNA gene but is added later as a post-transcriptional modification.
It was not known what is present in place of thymidine in TC loop and which adenine was
converted to 1-methyl adenine in M. smegmatis tRNAs. 1-methyl adenine is generally found in
tRNA from eukaryotes but not in prokaryotes. These issues were resolved by two approaches by
TR’s group in collaboration with Nishimura’s group. In one case they completely sequenced the
initiator tRNA of M. smegmatis. In the other, they developed a novel method to map the
modified bases within the tRNA structure starting with total tRNA.
Sequencing of Initiator tRNA
The complete sequence of transfer RNAs from many species is now known. The initiator tRNA
is charged with formylated methionine, which is the initiation codon for protein synthesis in
prokaryotes in almost all genes and this tRNA enters the P site on the ribosomes (Figure 1).
There are characteristic differences in initiator tRNA sequence between species. Therefore to
examine if the sequence of initiator tRNA of Mycobacteria is similar to that of eukaryotes or
eubacteria (true bacteria like E. coli) complete sequencing of tRNAfMet from M. smegamtis
was taken up. Around this time Nishimura’s group had developed a novel method for
sequencing tRNAs, that uniquely identified the modified nucleotides also while sequencing.
The major method used for identification of modified bases at that time was thin layer
chromatography (TLC) which took advantage of differential partition of nucleotides between a
mobile phase and a stationary phase, while sequencing was based on identifying the 5’end
nucleotide of fragments of tRNA fractionating based on size by electrophoresis. Kuchino in
Nishimura’s group ingeniously combined the two to read the sequence as well as the modification, if any, on the nucleotide. A limited cleavage of pure initiator tRNA by hot formamide
generates a single random cut on each tRNA molecule. These have a hydroxyl group at the 5’end
and therefore can accept radioactive phosphate group. When this pool of fragments is subjected
to electrophoresis they separate based on size and each one differs from the immediate smaller
fragment by one nucleotide. Each band is cut out from the gel and digested with RNAse P1 that
cleaves them to give 5’phosphate containing nucleosides. Then the mixture of nucleotide
monophosphates is separated by TLC. Each 5’end nucleotide can be identified as labeled
radioactive monophosphate, on exposure of the TLC plates to X-ray films. The advantage was
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that based on the position on the TLC plate one could not only identify all the normal
nucleotides but also the modified nucleotides unambiguously. In parallel the tRNA was
sequenced by a second method.
Reading the thesis where this is described, I realized that the  32P ATP that is required for the
exchange reaction was also synthesized in the lab while it is only a phone call/e-mail away now!
The sequence of initiator tRNAfMet of M. smegmatis obtained by this method is shown in
Figure 2. The features of having m1A as the 5th base and uridine in place of ribothymidine, and
also G.C, C.G, G.C as the base pairs at the 5’ end showed that initiator tRNA of M. smegmatis
shares its sequence features with Streptomyces [8]. It was interesting to know that m1A is
present in tRNA from other species of Mycobacteria including M. tuberculosis. TR’s group also
purified the enzyme that mediates this methylation [9]. With a desire to design new active
principles for potential therapeutic use against tuberculosis, an organic chemist Ramamurthy
joined TR’s group to synthesise analogues of adenine, coded as SIBA. When tested it was found
to have inhibitory activity on the formation of m1A. His group tried to analyse the functional
significance of this modification by measuring the efficiency of cross aminoacylation of E. coli
tRNA with and without m1A. Unfortunately some of these results remained unpublished.
After a gap of about two decades, in the Department of Microbiology and Cell Biology at IISc
where TR did all his work and also led the department as the Chairman for many years, Umesh
Varshney took up work on initiator tRNA and m1A tRNA methylase from M. tuberculosis. He
observed that there is a single copy of initiator tRNA gene in both M. smegmatis and M.
tuberculosis. In M. tuberculosis the CCA end is encoded in the gene while in M.smegmatis it is
added after transcription. Varshney’s group found that m1A tRNA methylase differs from that
of yeast not only in its subunit composition but also in biochemical properties. In yeast, knock
out of m1A tRNA methylase is lethal. Varshney et.al suggest that m1A methylase could be a
potential drug target. The crystal structure of the enzyme was also solved by Varshney and
coworkers.
Do Modified Bases Have Functional Significance?
Modified bases within or adjacent to the anticodon are important for filling in for codon
degeneracy. For example valine has three codons GUU, GUA and GUG in E. coli. Valyl tRNA
that contains Uridine-5-oxyacetic acid in the first position of the anticodon can recognize all
three codons while this property is lost when the modification is absent. Base modification
containing sulphur groups are known to confer thermal stability to tRNA from Thermus
thermophilus bacteria. More recently, it is found that tRNAs from tumour cells are less
modified than their counterparts from normal cells. Point mutation in one of the tRNALeu coding
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genes in mitochondria causes diseases called MELAs (mitochondrial myopathy, encephalopathy) and MERRF (a form of myoclonus epilepsy). The molecular basis of pathology in these
diseases is attributed to the absence of modified base in tRNALeu due to single nucleotide
variation in the gene.
Thus tRNA modifications appear to fine-tune protein production in cells. In spite of this,
targeting enzymes that modify bases in several tRNAs remains an unexplored area for drug
discovery specially for infectious diseases like tuberculosis. It is very gratifying personally for
me to have been associated with the work that TR carried out on modified nucleotides in tRNA,
though as a tail-ender in the long list of his illustrious students.
Suggested Reading
[1]
C de Duve, The second genetic code, Nature, Vol.333, p.117, 1988.
[2]
B R Vani, T Ramakrishnan, Y Taya, S Noguchi, Z Yamazumi and S Nishimura, Occurrence of 1methyladenosine and absence of ribothymidine in transfer ribonucleic acid of Mycobacterium smegmatis, J.
Bacteriology, Vol.137, No.3, pp.1084–1087, 1979.
[3]
[4]
K P Gopinathan, T Ramakrishnan (1922-2008) Personal News, Current Science, Vol.94, No.7. pp.935–936, 2008.
V Brahmachari, T Ramakrishnan, Modified bases in transfer RNA, J. Biosciences, Vol.6, No.5, pp.757–770,
1984.
[5]
P Schimmel, L R de Pouplana, Transfer RNA; From minihelix to genetic code, Cell, Vol.81, pp.983–986, 1995.
[6]
S Nishimura and K Watanabe, Discovery of modified nucleosides from the early days to the present: A personal
perspective, J. Biosciences, Vol.31, No.4. pp.465–475, 2006.
[7]
R Giege, Towards a more complete view of tRNA biology, Nature Structural and Molecular Biology, Vol.
15, No.10, pp.1007–1014, 2008.
[8]
B R Vani, T Ramakrishnan, Y Kuchino and S Nishimura, Nucleotide sequence of initiator tRNA from
Mycobacterium smegmatis, Nucleic Acids Research, Vol.12, No.9, pp.3933–3936, 1984.
[9]
V Brahmachari and T Ramakrishnan, Studies on 1-methyl adenine transfer RNA methyltransferase of Myco
bacterium semgmatis, Arch. Microbiology, Vol.140, pp.91–95, 1984.
Address for Correspondence
Vani Brahmachari, Dr B R Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110 007, India.
Email: [email protected]
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