Molecular Biology of Archaea

Our research interests are in the area of gene regulation in Archaea (Archaebacteria), with emphasis on RNA processing. Archaea are one of the three domains of life, the other two being Bacteria (eubacteria) and Eukarya (eukaryotes). Most of the Archaea grow under extreme environmental conditions. Archaea include methanogens (producing methane), extreme halophiles (growing in two molar to nearly saturated salt concentrations), thermophiles and hyperthermophiles (growing at 65 to 105 degrees C), acidophiles, alkaliphiles, etc. Archaea, like Bacteria, are prokaryotic in organization, but show similarities to several components of the eukaryotic replication, transcription and translation systems.

RNA Processing in Archaea

Posttranscriptional RNA processing involves additions and deletions at the ends of the transcripts, RNA splicing, RNA editing, residue modification, etc. Previously we have sequenced nearly complete set of tRNAs from a halophilic archaeon -- Haloferax volcanii (Halobacterium volcanii), characterized several modified nucleotides present in archaeal tRNAs, and reported the presence of introns in several archaeal tRNA genes. Our current research interests involve RNA splicing and sRNA-guided modification of RNA residues and their role in the biology of Archaea.

RNA Splicing in Archaea

Splicing of introns in Archaea involves an endonuclease and a ligase. We have established an in vitro system to study RNA splicing in halophilic Archaea. Using this system we have shown that during ligation, the phosphate at the splicing junction is derived from the precursor RNA. This is also the case for the animal type tRNA splicing ligase and is different from the tRNA splicing ligase of yeast. We have also shown that during splicing not only the two exons are ligated together but the introns are also ligated to form circular RNAs. In some cases these circular products are retained in the cell, suggesting that they may have a functional role.

Work from our laboratory (unpublished) and other laboratories has shown that ribosomal RNA processing in Archaea also involves splicing endonuclease and ligase activity.

                       (A)                                                                                   (B)
             
A model for the reactions occurring during RNA splicing in Archaea(A) Symmetric natures of the bulge-helix-bulge (BHB) containing endonuclease substrate and the two seven-base hairpin loops in the ligase products. Arrows indicate the splice sites in the substrate and the asterisks indicate the junction phosphates in the products.  (B) Reactions involving specific phosphodiester linkages during RNA splicing. 1–9 and 1*–9* are residues involved in the formation of BHB in the substrates and hairpins in the products. The two phosphates (p1 and p2) and 2‘, 3', and 5' positions involved in the reactions are indicated. (E1 and E2) two exons; (I) intron.

sRNAs (Small Nucleolar RNA like RNAs) in Archaea

The tRNAs and rRNAs of all organisms contain many different types of nucleotide modifications. In eukaryotes most of the 2'-O-methylation and pseudouridylation of rRNAs are carried out by snoRNPs (small nucleolar ribonucleoproteins), the RNA components of which function as guides to select the sites of modification. The 2'-O-methylations are carried out by box C/D RNAs along with their associated proteins. Homologs of box C/D snoRNAs and their associated proteins have been reported in Archaea. These are known as sRNAs. The intron of pre-tRNATrp of Haloferax volcanii has features of box C/D sRNA and is reported to guide 2’-O-methylation of targets in exons. Our earlier results have shown that, in vivo, residues at positions 34 and 39 of the mature tRNATrp of H. volcanii are 2'-O-methylated. We are investigating the role of this pre-tRNATrp intron as box C/D guide RNA. Positioning of the guide and the targets in the same precursor presented a situation, where it was tempting to speculate that the guide-target pairing can occur in cis to accomplish 2’-O-methylation of the residues at position 34 and 39. Such a phenomenon would be unique, since in all other cases of RNA-guided 2'-O-methylation reactions in eukaryotes and Archaea, guide and target regions are located on two separate RNAs .

Primary sequences and predicted secondary structures of the H. volcanii pre-tRNATrp. The tRNA anticodon sequence (CCA) is indicated in large letters in the pre-tRNA. The exon-intron junctions, designated by arrows, are located within the bulge-helix-bulge structure required for pre-tRNA splicing. Boxes C, D, C', and D' are enclosed and designated. Complementary guide and target sequences are designated by the thick lines (box C/D) and thin lines (C'/D'). The target nucleotides in the pre-tRNA are numbered C34 and U39 according to the standard tRNA numbering system. Complementary guide (lower case) and target (upper case) nucleotide pairs (g117:C34 and a70:U39) are indicated in black squares (C/D motif) and black circles (C'/D' motif), respectively.

We have developed a heterologous, in vitro modification system using Methanocaldococcus jannaschii proteins and pre-tRNATrp from H. volcanii. Using this system we have proven that modifications at positions 34 and 39 of the pre-tRNA occur through guide-target pairing in trans, not in cis. The intron as part of the pre-tRNA or in free linear or circular form, produced during splicing reaction can act as guide in these in vitro reactions. Using the same system we have also shown that methylation of the two nucleotides, guided by the two sites of a single box C/D RNA occur sequentially. Box C'/D' RNP-guided U39 methylation first requires box C/D RNP-guided methylation of C34. Based on these and earlier results (retention of circularized introns in vivo) we propose an in vivo scenario, where the intron splicing and RNA methylation events occur in concert.

Proposed model for the trans-2'-O-methylation of H. volcanii pre-tRNATrp in vivo. Thick arrows indicate the pre-tRNATrp processing pathway proceeding from transcription of the pre-tRNA through nucleotide methylation and splicing to the production of tRNATrp and excised intron. RNP assembly is denoted by thin arrows, and nucleotide modification guided intermolecularly by the intron-encoded box C/D RNP is denoted by dashed arrows.

Selected publication ( Complete list )

• Singh, S. K., P. Gurha, E. J. Tran, E. S. Maxwell and R. Gupta. 2004. Sequential 2'-O-methylation of Archaeal pre- tRNATrp nucleotides is guided by the intron-encoded but trans-acting box C/D ribonucleoprotein of pre- tRNA. J. Biol. Chem. 279: 47661-47671.
  (Pubmed)

• Salgia , S. R., S. K. Singh, P. Gurha and R. Gupta. 2003. Two reactions of Haloferax volcanii RNA splicing enzymes: Joining of exons and circulrization of introns. RNA 9: 319-330.
  (Pubmed)

• Zofallova , L., Y. Guo and R. Gupta. 2000. Junction phosphate is derived from the precursor in the tRNA spliced by the archaeon Haloferax volcanii cell extract. RNA 6:1019-1030.
  (Pubmed)

• Gomes, I. and R. Gupta. 1997. RNA splicing ligase activity in the archaeon Haloferax volcanii. Biochem. Biophys. Res. Commun. 237:588-594.
  (Pubmed)

• Datta , P. K., L. K. Hawkins and R. Gupta. 1989. Presence of an intron in elongator methionine - tRNA of Halobacterium volcanii. Can. J. Microbiol. 35:189-194.
  (Pubmed)

• Gupta, R. 1986. Transfer RNAs of Halobacterium volcanii: Sequences of five leucine and three serine tRNAs. System. Appl. Microbiol. 7:102-105.

• Daniels, C. J., R. Gupta and W. F. Doolittle. 1985. Transcription and excision of a large intron in the tRNA Trp gene of an archaebacterium, Halobacterium volcanii. J. Biol. Chem. 260:3132-3134.
  (Pubmed)

• Gupta, R. 1984. Halobacterium volcanii tRNAs: Identification of 41 tRNAs covering all amino acids, and the sequences of 33 class I tRNAs. J. Biol. Chem. 259:9461-9471.
  (Pubmed)

• Kaine , B. P., R. Gupta and C. R. Woese. 1983. Putative introns in tRNA genes of prokaryotes. Proc. Natl. Acad. Sci. USA 80:3309-3312.
  (Pubmed)

• Gupta, R., J. M. Lanter and C. R. Woese. 1983. Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium. Science 221:656-659.

• Gupta, R. and C. R. Woese. 1980. Unusual modification patterns in the transfer ribonucleic acids of archaebacteria. Curr. Microbiol. 4:245-249.