Expression of a yeast intron-containing tRNA in the archaeon Haloferax volcanii

Palmer, John R.; Nieuwlandt, D T; Daniels, Charles J.

doi:10.1128/jb.176.12.3820-3823.1994

Cited by 20 publications

(19 citation statements)

References 21 publications

(26 reference statements)

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“…We have previously shown that a 48-bp DNA fragment originating from the 5Ј region of the H. volcanii tRNA Lys gene is capable of directing efficient and precise transcription when introduced into these cells on plasmid pWL201 (39). Using the archaeal box A consensus (41,47,48,51) as a guide, combined with the observation that box A elements are generally located approximately 25 bp 5Ј of the transcription initiation site, we identified the sequence 5Ј-ATTTTACCCAC-3Ј as a potential box A element within the tRNA Lys DNA fragment.…”

Section: Resultsmentioning

confidence: 99%

“…1A). Transcription from this promoter begins within the expected box B element and results in the production of a single stable transcript that can be detected by Northern (RNA) analysis (37,39).…”

Section: Methodsmentioning

confidence: 99%

“…For example, the oligonucleotide MUTP-2 (5Ј-CGTGCAAAGCTTGAAAG GAAAGTC[G/T/C]TTTTACCCAC-3Ј) was used to introduce mutations at position Ϫ29. The plasmid pUC302, which contains the shortened H. volcanii tRNA Lys promoter immediately upstream of the yeast reporter gene (39), was used as the template DNA for PCR-based mutagenesis. The DNA between the regions of pUC302 complementary to each MUTP oligonucleotide and the universal pUC/M13 Ϫ40 primer was amplified by PCR.…”

Section: Methodsmentioning

confidence: 99%

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In vivo definition of an archaeal promoter

1995

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We have used a plasmid-based transcriptional reporter system to examine the transcriptional effects of 33 single point mutations in the box A region (TATA-like sequence) of the Haloferax volcanii tRNA Lys promoter. The most pronounced effects on transcriptional efficiency were found when the nucleotides corresponding to the TATA-like region were altered. Promoters with wild-type or higher levels of transcriptional activity conformed to the general archaeal box A consensus, 5-T/CTTAT/AA-3. The preference for a pyrimidine residue in the 5 position of this region and the exclusion of guanine and cytosine in the next four positions in the 3 direction are defining characteristics shared by all efficient archaeal promoters. We have also observed that replacement of a 10-nucleotide purine-rich sequence, located 5 of the H. volcanii tRNA Lys box A element, completely abolished transcription from this promoter. These data show that the H. volcanii tRNA Lys promoter is dependent on two separate, and essential, sequence elements. The possible functions of these sequences, in view of the recent descriptions of eucaryal-like transcription factors for Archaea, are discussed.The transcriptional apparatus of archaea (formerly archaebacteria [49]) bears a striking resemblance to the transcriptional systems found in eucarya (eucaryotes) (for a review, see reference 51). Unlike the relatively simple bacterial RNA polymerases, the archaeal and the eucaryal RNA polymerases display complex subunit compositions. Archaeal RNA polymerases typically contain 12 or more subunits, and these proteins share immunological cross-reactivity and sequence similarity with eucaryal RNA polymerase subunits (51). The relatedness of these two systems can also be seen in the structures of their promoter elements. A comparison of sequences upstream of archaeal genes has revealed two conserved sequence elements, box A and box B (17,42,48,51). The box A element is located approximately 25 bp upstream of the transcription start site and has the general consensus 5Ј-T/CTTAT/ AA-3Ј. The core TATA sequence of this element is similar in location and sequence to the TATA promoter element recognized by eucaryal RNA polymerase II (5, 41, 47). The box B element contains the transcription start site and has a weak consensus 5Ј-T/CG/A-3Ј, with transcription beginning with the purine residue (17). Functional studies using in vitro transcription systems from Methanococcus vannielii, Methanococcus thermolithotrophicus, and Methanobacterium thermoautotrophicum (13, 14a, 18, 19, 25a) and Sulfolobus shibatae (17, 21a) have confirmed that the core box A TATA-like sequence is important in determining the efficiency of transcription initiation. The archaeal and eucaryal transcription systems may also share common mechanisms for promoter recognition and transcription initiation. In its most general form, initiation of transcription by eucaryal RNA polymerases is dependent on the interaction of the core RNA polymerase with proteins, transcription factors, and regulatory protei...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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In vivo definition of an archaeal promoter

1995

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“…The cct1 and cct2 5Ј flanking sequences were cloned into the plasmid pWL201 expression vector (39) as HindIII-XbaI fragments. In the case of cct1, whose 5Ј flanking region contained a HindIII endonuclease restriction site, the putative core promoter region was first cloned into the HindIII and XbaI sites of pWL201, and then the remaining upstream sequence was ligated into the expression construct as a HindIII-HindIII fragment.…”

Section: Methodsmentioning

confidence: 99%

Characterization of two heat shock genes from Haloferax volcanii: a model system for transcription regulation in the Archaea

et al. 1997

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The expression of two heat-responsive cct (chaperonin-containing Tcp-1) genes from the archaeon Haloferax volcanii was investigated at the transcription level. The cct1 and cct2 genes, which encode proteins of 560 and 557 amino acids, respectively, were identified on cosmid clones of an H. volcanii genomic library and subsequently sequenced. The deduced amino acid sequences of these genes exhibited a high degree of similarity to other archaeal and eucaryal cct family members. Expression of the cct genes was characterized in detail for the purpose of developing a model for studying transcription regulation in the domain Archaea. Northern (RNA) analysis demonstrated that the cct mRNAs were maximally induced after heat shock from 37 to 55°C and showed significant heat inducibility after 30 min at 60°C. Transcription of cct mRNAs was also stimulated in response to dilute salt concentrations. Transcriptional analysis of cct promoter regions coupled to a yeast tRNA reporter gene demonstrated that 5 flanking sequences up to position ؊233 (cct1) and position ؊170 (cct2) were sufficient for promoting heat-induced transcription. Transcript analysis indicated that both basal transcription and stress-induced transcription of the H. volcanii cct genes were directed by a conserved archaeal consensus TATA motif (5-TTTATA-3) centered at ؊25 relative to the mapped initiation site. Comparison of the cct promoter regions also revealed a striking degree of sequence conservation immediately 5 and 3 of the TATA element.All living organisms adapt to adverse environmental conditions by evolving specific molecular responses. In particular, the universally conserved heat shock response occurs when cells are exposed to elevated temperatures, resulting in the rapid and transient overproduction of a limited class of proteins called the heat shock proteins (HSPs). The HSPs produced in the domains Bacteria and Eucarya are highly conserved both in structure and function, and their induction is generally regulated at the transcription initiation level (recently reviewed in reference 31).Among the HSPs, the Cct family is a recently identified group of molecular chaperonins that are distinct from members of the well-studied Hsp60/GroEL family. So far, members of the Cct family have been identified only in the domains Archaea and Eucarya, where they are known variously as Tcomplex polypeptide, or TCP (15); chaperonin-containing Tcp-1, or Cct (25, 62); TCP-1 ring complex, or TriC (13); thermosome complex (59); and thermophilic factor-55, or TF55 (54). Protein members of the Cct and Hsp60/GroEL families display similar double-ringed toroidal structures as their active forms. Differences between the two families include the absence of an HSP10/GroES-like accessory protein necessary for Cct function (25) and the hetero-oligomeric structure of Cct complexes, which contain either 2 (58) or 8 to 10 different subunits (61). Among the members of the Archaea, members of the Cct protein family have been documented in many hyperthermophiles, including Sulfolo...

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“…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)(7)(8). By contrast, the eukaryal enzyme normally acts in a mature-domain-dependent mode (3,4).…”

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The dawn of dominance by the mature domain in tRNA splicing

Tocchini-Valentini

Fruscoloni

Tocchini‐Valentini

2007

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

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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...

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Expression of a yeast intron-containing tRNA in the archaeon Haloferax volcanii

Cited by 20 publications

References 21 publications

In vivo definition of an archaeal promoter

In vivo definition of an archaeal promoter

Characterization of two heat shock genes from Haloferax volcanii: a model system for transcription regulation in the Archaea

The dawn of dominance by the mature domain in tRNA splicing

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