Initiator transfer RNAs

RajBhandary, Uttam L.

doi:10.1128/jb.176.3.547-552.1994

Cited by 134 publications

(135 citation statements)

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“…In eubacteria, a mismatch in the distal pair in the acceptor stem (involving the Y-terminal nucleotide) is one of the determinants that precludes initiator tRNA from also being used in elongation. Mutant eubacterial tRNAs that have restored base pairing at the distal end of the acceptor stem are able to function in both initiation and elongation [34]. Because the T. pyriformis mitochondrial tRNA Met also has a normal base pair at the distal end of the acceptor stem (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…3) has three adjacent G-C base pairs at the base of the anticodon stem that are known to be important for binding of eubacterial initiator tRNA to the P site of the ribosome [34]. In eubacteria, a mismatch in the distal pair in the acceptor stem (involving the Y-terminal nucleotide) is one of the determinants that precludes initiator tRNA from also being used in elongation.…”

Section: Discussionmentioning

confidence: 99%

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Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNA^Met from Tetrahymena pyriformis

1995

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We have now isolated and characterized the smallest T. pyriformis mitochondrial tRNA and we show here that it has the primary sequence that we had predicted from the trnM sequence. The transcript also contains a Y-terminal CCA sequence, not encoded in the mtDNA, as well as several modified nucleosides characteristic of known functional tRNAs. Materials and methodsT. pyriformis, amicronucleate strain ST (obtained from Y. Suyama, Department of Biology, University of Pennsylvania), was grown at 28°C with constant shaking in 500 ml NeWs medium [9]. The culture was chilled on ice for 10 15 min, following which cells were collected by centrifugation at 2000 rpm for 5 min, resuspended, and centrifuged at 2000 rpm for 5 min through a layer of ice-cold homogenizing medium (0.35 M sucrose, 10 mM Tris-HC1, pH 7.2, 2 mM EDTA) [10]. Resuspended cells in 50 ml homogenizing medium were disrupted by three passages through a hand emulsifier (nozzle unscrewed 2.5 turns) [11]. Unbroken cells and debris were removed by centrifugation at 3000 rpm (IEC) for 5 min. A mitochondrial pellet was recovered from the resulting supernatant by centrifugation at 8000 rpm for 5 min and washed twice by resuspension in 50 ml homogenizing medium followed by centrifugation (5 min, 8000 rpm). The final mitochondrial pellet was resuspended in 10 ml 0.15 M NaC1, 0.1 M EDTA (pH 9.0) [12] at room temperature. Ten ml of 4% SDS in the same buffer were added and the mitochondrial lysate was then extracted three times with phenol/cresol [13] at room temperature. Nucleic acids were precipitated by addition of 2 vols. of 95% ethanol. For preparation of a post-ribosomal supernatant [14], purified mitochondria were resuspended in buffer (100 mM KC1, 10 mM MgCI 2, 10 mM Tris-HCl, pH 7.4) and lysed at a final concentration of 2% Triton X-100. After removal of membrane fragments by centrifugation at 10,000 rpm for 10 rain (IEC), the clarified mitochondrial lysate was centrifuged at 42,000 rpm for 90 min in a Ti50 rotor to sediment ribosomes. Transfer RNA was isolated from the supernatant by detergent phenol/cresol extraction.Nucleic acid samples were either used directly for 3'-end-labeling or, alternatively, the RNA samples (5 ~tg post-ribosomal supernatant RNA or 20/lg total nucleic acid) were dissolved in 7.5/11 NMF/urea loading buffer [15] containing 0.1% (w/v) xylene cyanol or Bromophenol blue and heated to 65°C for 5 min. After electrophoresis in a thin (0.5 ram) 6% polyacrylamide gel containing 7 M urea, the smallest tRNA was visualized by ethidium bromide staining and eluted [16] in the presence of 20 gtg linear polyacrylamide carrier (prepared as in [17], extracted with phenol, precipitated with ethanol and re-dissolved at 2.5 flg//.tl). The unlabeled tRNA was either re-purified by gel-electrophoresis and then subjected to modified nucleotide analysis [16] or used directly for end-labeling. 3'-End-labeled tRNA [18] was purified by gel-electrophoresis and used for chemical sequence analysis [18]; both 3'-and 5'-endlabeled tRNAs were subjected to enzymatic s...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNA^Met from Tetrahymena pyriformis

1995

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“…The other trait used to reach similar conclusions is the Met-tRNA fMet ->fMet-tRNA fMet pathway (Rajbhandary, 1994). This pathway should be an ancestral trait because it is one of the five known pathways on tRNAs representing the principal manifestation of the coevolution theory of genetic code origin (Wong, 1975(Wong, , 2005Di Giulio, 1999b, 2008b.…”

mentioning

confidence: 94%

“…These arguments have used two traits: the tRNA split genes of Nanoarchaeum equitans and the Met-tRNA fMet ->fMet-tRNA fMet pathway (Randau et al, 2005;Rajbhandary, 1994). Based on the hypothesis that a hairpin structure was the precursor of the tRNA molecule and that the assembly of two hairpin structures might have created this molecule, a theory was constructed predicting the existence of mini-genes codifying for these hairpin structures and, thus, for half tRNAs (Di Giulio, 1992, 1995, 1999a, 2006a, b, c, 2009a.…”

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confidence: 99%

“…This pathway is present only in the Bacteria domain (Rajbhandary, 1994) which, together with the differences existing between the three domains of life as far as the initiation of protein synthesis is concerned, lead us to believe that this pathway originated very late on in the evolution of the genetic code and only when the line of divergence leading to Bacteria was defined (Di Giulio, 2001, 2010. It must also be believed that when the phyletic pathway leading to Bacteria was defined, not only was the protein synthesis initiation apparatus still rapidly evolving (for the very reason that the Met-tRNA fMet ->fMet-tRNA fMet pathway is present only in the Bacteria domain) but also, and more generally, the entire translation apparatus must have been in rapid and progressive Darwinian evolution (Di Giulio, 2001, 2010.…”

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Biological evidence against the panspermia theory

Giulio

2010

Journal of Theoretical Biology

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InitiatortRNAsin Bacteria and Eukaryotes

Rasmussen

Laursen²,

Mortensen

et al. 2009

Encyclopedia of Life Sciences

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A special type of transfer ribonucleic acid (tRNA) is employed in the initiation step of protein synthesis. The differences between the tRNAs involved in initiation and elongation establish the specificity and recognition of the tRNAs by the factors and enzymes involved in the initiation and elongation steps of protein synthesis. The important determinants of the initiator tRNA in bacteria are the absence of a Watson–Crick base pair between positions 1 and 72 in the acceptor stem and the presence of three conserved consecutive G:C base pairs in the anticodon stem. Formylation of the methionine attached to this initiator tRNA is an important feature. Conversely, the eukaryotic initiator tRNA is mainly determined by the presence of a particular A1:U72 Watson–Crick base pair in the acceptor stem as well as the nature of the base pairs 50:64 and 51:63 in the TΨC (T) stem. Key concepts Initiator tRNA brings methionine to the initiation complex for initiation of protein synthesis. Structural features of initiator tRNAs ensure that they are recognized by translation initiation factors and discriminated against by translation elongation factors. The important determinants of initiator tRNA in bacteria are the absence of a Watson–Crick base pair between positions 1 and 72 in the acceptor stem and the presence of three conserved consecutive G:C base pairs in the anticodon stem. The important determinants of initiator tRNA in eukaryotes are the presence of a particular A1:U72 Watson–Crick base pair in the acceptor stem as well as the nature of the base pairs 50:64 and 51:63 in the T stem. Bacterial initiator tRNA is aminoacylated by methionyl tRNA synthetase, which mainly interacts with the anticodon. The methionine of bacterial initiator tRNA is formylated by methionyl tRNA transformylase, which mainly recognizes the absence of the 1:72 base pair. Initiation factor IF2 ensures recognition and correct binding of bacterial initiator tRNA to the ribosomal P‐site by interacting primarily with the formyl group and the 3′ end of the acceptor arm. Initiation factor IF3 performs proofreading of the 30S initiation complex by verifying base pairing between 5′C of bacterial initiator tRNA anticodon and 3′G of mRNA initiation codon. Bacterial initiator tRNA undergoes several conformational changes during translation initiation to ensure correct positioning in the P‐site of the ribosome. Bacterial initiator tRNA is not a substrate for peptidyl tRNA hydrolase.

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Initiator transfer RNAs

Cited by 134 publications

References 39 publications

Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNA^Met from Tetrahymena pyriformis

Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNA^Met from Tetrahymena pyriformis

Biological evidence against the panspermia theory

InitiatortRNAsin Bacteria and Eukaryotes

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Initiator transfer RNAs

Cited by 134 publications

References 39 publications

Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNAMet from Tetrahymena pyriformis

Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNAMet from Tetrahymena pyriformis

Biological evidence against the panspermia theory

InitiatortRNAsin Bacteria and Eukaryotes

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Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNA^Met from Tetrahymena pyriformis

Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNA^Met from Tetrahymena pyriformis