Conservation of Substrate Recognition Mechanisms by tRNA Splicing Endonucleases

Fabbri, S; Fruscoloni, Paolo; Bufardeci, E; Nicola-Negri, Elisa Di; Baldi, M.I.; Attardi, Domenica Gandini; Mattoccia, Emilio; Tocchini‐Valentini, Glauco P.

doi:10.1126/science.280.5361.284

Cited by 61 publications

(75 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To determine whether the ␣ SULSO enzyme has a dual functional role like the METJA monomer, we tested the purified ␣ SULSO protein for cleavage of pre-tRNA archaeuka. We already knew that the intron of this substrate could be precisely excised by using a partially purified extract from SULSO (12). Fig.…”

Section: Resultsmentioning

confidence: 99%

“…3A), a synthetic molecule containing an intron recognized by either archaeal or eukaryotic endonucleases (12,20). Two truncated forms of the ARCFU enzyme, NR ARCFU and CR ARCFU, were generated.…”

Section: Resultsmentioning

confidence: 99%

“…In Vitro Transcription. The DNA template was prepared and transcribed by T7 RNA polymerase (12)(13)(14) using the T7-Megashort-script kit (Ambion, Austin, TX). Transcripts were internally labeled with [ 32 P]UTP [800 Ci͞mmol (1 Ci ϭ 37 GBq), Amersham Pharmacia].…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Structure, function, and evolution of the tRNA endonucleases of Archaea: An example of subfunctionalization

Tocchini-Valentini

Fruscoloni

Tocchini‐Valentini

2005

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

106

View full text Add to dashboard Cite

We have detected two paralogs of the tRNA endonuclease gene of Methanocaldococcus jannaschii in the genome of the crenarchaeote Sulfolobus solfataricus. This finding has led to the discovery of a previously unrecognized oligomeric form of the enzyme. The two genes code for two different subunits, both of which are required for cleavage of the pre-tRNA substrate. Thus, there are now three forms of tRNA endonuclease in the Archaea: a homotetramer in some Euryarchaea, a homodimer in other Euryarchaea, and a heterotetramer in the Crenarchaea and the Nanoarchaea. The last-named enzyme, arising most likely by gene duplication and subsequent ''subfunctionalization,'' requires the products of both genes to be active. molecular evolution ͉ RNA-protein interactions ͉ tRNA splicing G ene duplication is the primary source of new genes. The ''subfunctionalization'' hypothesis argues that duplicate genes experience degenerate mutations that divide the activity encoded by a single ancestral gene among its descendants (1). Here, we report a striking example of subfunctionalization.In Archaea, the tRNA endonuclease plays a key role in assuring the correct removal of the intron from pre-tRNAs and pre-rRNA (2-6), which constitute the core of the translation machinery. Crystal structures of the tRNA endonucleases from Methanocaldococcus jannaschii (METJA) and Archaeoglobus fulgidus (ARCFU), both belonging to the phylum Euryarchaeota, are available (7,8). These structures differ in a remarkable way. The structure of the homotetrameric endonuclease from METJA reveals two different functional roles for the monomeric units. The METJA endonuclease is organized as a dimer of dimers, with one subunit from each dimer participating in the catalytic cleavage reaction (the catalytic subunit) and the other (structural) subunit acting to place the two catalytic subunits correctly in space.The crystal structure of the ARCFU endonuclease, by contrast, shows it to be a homodimer. Each subunit contains two similar repeating domains that are homologous to the subunit structure of the homotetrameric enzyme from METJA; the C-terminal repeat (CR) is the active domain, and the N-terminal repeat (NR) acts to stabilize the dimer.The overall shape and size of the homodimeric ARCFU endonuclease resembles that of the homotetrameric METJA enzyme.Both METJA and ARCFU belong to the Euryarchaeota. Nothing is known about the tRNA endonuclease of Crenarchaeota, the other main family of Archaea. To determine the properties of a crenarchaeal endonuclease, we searched the genome sequence of Sulfolobus solfataricus (SULSO) for homologs of the METJA endonuclease and found two candidate sequences.Characterization of the two gene products reveals that both are required for tRNA endonuclease activity, each presumably functioning like one-half of the ARCFU enzyme. Detailed analysis of the amino acid sequences of the two proteins supports the idea that they evolved by the process called subfunctionalization (1, 9, 10). Materials and MethodsIdentification of the Homologs. Th...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Structure, function, and evolution of the tRNA endonucleases of Archaea: An example of subfunctionalization

Tocchini-Valentini

Fruscoloni

Tocchini‐Valentini

2005

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

106

View full text Add to dashboard Cite

show abstract

“…This hybrid pre-tRNA is analogous, in its structure, to other chimeras we used in in vitro experiments, such as the pre-tRNA ArchEuka Phe . As we have shown previously, pre-tRNA ArchEuka Phe is cleaved by either the eukaryal or the archaeal endonuclease (Fabbri et al 1998).…”

Section: Sup4 Pre-trna Archeuka Tyr Is Cleaved In Vitromentioning

confidence: 94%

A pre-tRNA carrying intron features typical of Archaea is spliced in yeast

Segni¹,

Borghese²,

Sebastiani³

et al. 2004

RNA

Self Cite

View full text Add to dashboard Cite

Archaeal pre-tRNAs are characterized by the presence of the bulge-helix-bulge (BHB) structure in the intron stem-and-loop region. A chimeric pre-tRNA was constructed bearing an intron of the archaeal type and the mature domain of the Saccharomyces cerevisiae suppressor SUP4 tRNA Tyr . This pre-tRNA ArchEuka is correctly cleaved in several cell-free extracts and by purified splicing endonucleases. It is also cleaved and ligated in S. cerevisiae cells, providing efficient suppression of nonsense mutations in various genes.

show abstract

“…There is, however, a single striking exception: a specific structure consisting of two 3-nt bulges separated by a 4-bp helix, the so called bulge-helix-bulge (BHB) motif, has been shown to be recognized and cleaved in vitro by the eukaryal enzyme without any reference to the mature domain (12,13). This finding presents interesting evolutionary implications.…”

mentioning

confidence: 87%

The dawn of dominance by the mature domain in tRNA splicing

Tocchini-Valentini

Fruscoloni

Tocchini‐Valentini

2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

The relationship between enzyme architecture and substrate specificity among archaeal pre-tRNA splicing endonucleases has been investigated more deeply, by using biochemical assays and model building. The enzyme from Archeoglobus fulgidus (AF) is particularly interesting: it cleaves the bulge-helix-bulge target without requiring the mature tRNA domain, but, when the target is a bulge-helix-loop, the mature domain is required. A model of AF based on its electrostatic potential shows three polar patches interacting with the pre-tRNA substrate. A simple deletion mutant of the AF endonuclease lacking two of the three polar patches no longer cleaves the bulge-helix-loop substrate with or without the mature domain. This single deletion shows a possible path for the evolution of eukaryal splicing endonucleases from the archaeal enzyme. molecular evolution ͉ RNA-protein interactions ͉ tRNA endonucleases A ccuracy in tRNA splicing is essential for the formation of functional tRNAs and therefore for gene expression. In Bacteria, pre-tRNA introns are self-splicing group I introns, and the splicing mechanism is autocatalytic. In Eukarya, tRNA introns are small and invariably interrupt the anticodon loop one base 3Ј to the anticodon. In Archaea, the introns are also small and often reside in the same location as eukaryal tRNA introns, but not always (1, 2). In both Eukarya and Archaea, the specificity for recognition of the pre-tRNA resides in the endonucleases (3-5). These enzymes remove the intron by making two independent endonucleolytic cleavages. It is generally accepted that the archaeal enzymes act without any reference to the mature domain but instead recognize specific structures that define the intron-exon boundaries (6-8). By contrast, the eukaryal enzyme normally acts in a mature-domain-dependent mode (3, 4).The exact way in which the eukaryal endonucleases recognize the precursors has yet to be determined, but many RNA-protein interactions are presumably required. Previous studies have shown the role of the three-dimensional structure and of specific invariant bases of the mature domain in recognition and binding by the enzyme (3, 4). The importance of structural regularities in the precursors suggested an anchor-and-measure mechanism in which the endonuclease binds to one or more reference sites in the mature domain, which are common to all pre-tRNAs, and measures the distance to the equivalently positioned intron-exon junctions. This hypothesis was supported by experiments that involved the engineering of changes in the distance that separate the mature domain from the splice sites. These alterations changed the size of the intron in a predictable way: the insertion of 1 bp in the anticodon stem increased the size of the intron by two bases, one at each end, and the insertion of 2 bp in the anticodon stem increased the size of the intron by 4 nt (3, 4). These striking results, which had been observed in specific instances, were generalized and taken to mean that there are no important recognition elements at t...

show abstract

Conservation of Substrate Recognition Mechanisms by tRNA Splicing Endonucleases

Cited by 61 publications

References 7 publications

Structure, function, and evolution of the tRNA endonucleases of Archaea: An example of subfunctionalization

Structure, function, and evolution of the tRNA endonucleases of Archaea: An example of subfunctionalization

A pre-tRNA carrying intron features typical of Archaea is spliced in yeast

The dawn of dominance by the mature domain in tRNA splicing

Contact Info

Product

Resources

About