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At least 19 recordsLinked to original sources
PubMed2001

New insight into RNase P RNA structure from comparative analysis of the archaeal RNA.

A detailed comparative analysis of archaeal RNase P RNA structure and a comparison of the resulting structural information with that of the bacterial RNA reveals that the archaeal RNase P RNAs are strikingly similar to those of Bacteria. The differences between the secondary structure models of archaeal and bacterial RNase P RNA have largely disappeared, and even variation in the sequence and structure of the RNAs are similar in extent and type. The structure of the cruciform (P7-11) has been reevaluated on the basis of a total of 321 bacterial and archaeal sequences, leading to a model for the structure of this region of the RNA that includes an extension to P11 that consistently organizes the cruciform and adjacent highly-conserved sequences.

Base Sequence↗
PubMed2004

Transcription by an archaeal RNA polymerase is slowed but not blocked by an archaeal nucleosome.

Archaeal RNA polymerases (RNAPs) are closely related to eukaryotic RNAPs, and in Euryarchaea, genomic DNA is wrapped and compacted by histones into archaeal nucleosomes. In eukaryotes, transcription of DNA bound into nucleosomes is facilitated by histone tail modifications and chromatin remodeling complexes, but archaeal histones do not have histone tails and archaeal genome sequences provide no evidence for archaeal homologs of eukaryotic chromatin remodeling complexes. We have therefore investigated the ability of an archaeal RNAP, purified from Methanothermobacter thermautotrophicus, to transcribe DNA bound into an archaeal nucleosome by HMtA2, an archaeal histone from M. thermautotrophicus. To do so, we constructed a template that allows transcript elongation to be separated from transcription initiation, on which archaeal nucleosome assembly is positioned downstream from the site of transcription initiation. At 58 degrees C, in the absence of an archaeal nucleosome, M. thermautotrophicus RNAP transcribed this template DNA at a rate of approximately 20 nucleotides per second. With an archaeal nucleosome present, transcript elongation was slowed but not blocked, with transcription pausing at sites before and within the archaeal nucleosome. With additional HMtA2 binding, complexes were obtained that also incorporated the upstream regulatory region. This inhibited transcription presumably by preventing archaeal TATA-box binding protein, general transcription factor TFB, and RNAP access and thus inhibiting transcription initiation.

Archaeal Proteins↗
PubMed2000

Archaeal RNA polymerase subunits F and P are bona fide homologs of eukaryotic RPB4 and RPB12.

The archaeal and eukaryotic evolutionary domains diverged from each other approximately 2 billion years ago, but many of the core components of their transcriptional and translational machineries still display a readily recognizable degree of similarity in their primary structures. The F and P subunits present in archaeal RNA polymerases were only recently identified in a purified archaeal RNA polymerase preparation and, on the basis of localized sequence homologies, tentatively identified as archaeal versions of the eukaryotic RPB4 and RPB12 RNA polymerase subunits, respectively. We prepared recombinant versions of the F and P subunits from Methanococcus jannaschii and used them in in vitro and in vivo protein interaction assays to demonstrate that they interact with other archaeal subunits in a manner predicted from their eukaryotic counterparts. The overall structural conservation of the M. jannaschii F subunit, although not readily recognizable on the primary amino acid sequence level, is sufficiently high to allow the formation of an archaeal-human F-RPB7 hybrid complex.

Amino Acid Sequence↗
PubMed2000

Transcription factor S, a cleavage induction factor of the archaeal RNA polymerase.

We have analyzed the function of an archaeal protein (now called transcription factor S (TFS)) that shows sequence similarity to eukaryotic transcription factor IIS (TFIIS) as well as to small subunits of eukaryotic RNA polymerases I (A12.6), II (B12.2), and III (C11). Western blot analysis with antibodies against recombinant TFS demonstrated that this protein is not a subunit of the RNA polymerase. In vitro transcription experiments with paused elongation complexes at position +25 showed that TFS is able to induce cleavage activity in the archaeal RNA polymerase in a similar manner to TFIIS. In the presence of TFS, the cleavage activity of the RNA polymerase truncates the RNA back to position +15 by releasing mainly dinucleotides from the 3'-end of the nascent RNA. Furthermore, TFS reduces the amount of non-chaseable elongation complexes at position +25 as well as position +45. These findings clearly demonstrate that this protein has a similar function to eukaryotic TFIIS.

Amino Acid Sequence↗
PubMed2003

Analysis of the open region and of DNA-protein contacts of archaeal RNA polymerase transcription complexes during transition from initiation to elongation.

The archaeal transcriptional machinery is polymerase II (pol II)-like but does not require ATP or TFIIH for open complex formation. We have used enzymatic and chemical probes to follow the movement of Pyrococcus RNA polymerase (RNAP) along the glutamate dehydrogenase gene during transcription initiation and transition to elongation. RNAP was stalled between registers +5 and +20 using C-minus cassettes. The upstream edge of RNAP was in close contact with the archaeal transcription factors TATA box-binding protein/transcription factor B in complexes stalled at position +5. Movement of the downstream edge of the RNAP was not detected by exonuclease III footprinting until register +8. A first structural transition characterized by movement of the upstream edge of RNAP was observed at registers +6/+7. A major transition was observed at registers +10/+11. In complexes stalled at these positions also the downstream edge of RNA polymerase started translocation, and reclosure of the initially open complex occurred indicating promoter clearance. Between registers +11 and +20 both RNAP and transcription bubble moved synchronously with RNA synthesis. The distance of the catalytic center to the front edge of the exo III footprint was approximately 12 nucleotides in all registers. The size of the RNA-DNA hybrid in an early archaeal elongation complex was estimated between 9 and 12 nucleotides. For complexes stalled between positions +10 and +20 the size of the transcription bubble was around 17 nucleotides. This study shows characteristic mechanistic properties of the archaeal system and also similarities to prokaryotic RNAP and pol II.

Archaeal Proteins↗
PubMed2005

Determinants of transcription initiation by archaeal RNA polymerase.

Transcription in Archaea is catalyzed by an RNA polymerase that is most similar to eukaryotic RNA polymerases both in subunit composition and in transcription initiation factor requirements. Recent studies on archaeal transcription in diverse members of this domain have contributed new details concerning the functions of promoters and transcription factors in guiding initiation by RNA polymerase, and phylogenetic arguments have allowed modeling of archaeal transcription initiation complexes by comparison with recently described models of eukaryotic and bacterial transcription initiation complexes. Important new advances in reconstitution of archaeal transcription complexes from fully recombinant components is permitting testing of hypotheses derived from and informed by these structural models, and will help bring the study of archaeal transcription to the levels of understanding currently enjoyed by bacterial and eukaryotic RNA polymerase II transcription.

Amino Acid Sequence↗
PubMed2005

Archaeal RNA polymerase is sensitive to intrinsic termination directed by transcribed and remote sequences.

Archaea are prokaryotes with a single DNA-dependent RNA polymerase (RNAP) that is homologous to, and likely resembles the ancestor of all three eukaryotic RNAPs. In vitro studies have confirmed that initiation by archaeal RNAPs resembles the Pol II system, and we report the first detailed in vitro investigation of archaeal transcription termination. Methanothermobacter thermautotrophicus (M.t.) RNAP is susceptible to intrinsic termination at an intergenic sequence that conforms to a bacterial intrinsic terminator, as well as at bona fide bacterial intrinsic terminators. In contrast to bacterial RNAPs, M.t. RNAP also terminated in response to synthetic and natural oligo-T-rich sequences that were not preceded by sequences with any recognizable potential to form a stable RNA hairpin. Both template topology and temperature influenced the position and extent of termination in vitro, and the results argue that transcription of an upstream sequence can alter the termination response of the archaeal RNAP at a remote downstream sequence.

Archaeal Proteins↗
PubMed1995

Functional interaction of yeast and human TATA-binding proteins with an archaeal RNA polymerase and promoter.

TATA boxes are common structural features of eucaryal class II and archaeal promoters. In addition, a gene encoding a polypeptide with sequence similarity to eucaryal TATA-binding protein (TBP) has recently been detected in Archaea, but its relationship to the archaeal transcription factors A (aTFA) and B (aTFB) was unclear. Here, we demonstrate that yeast and human TBP can substitute for aTFB in a Methanococcus-derived archaeal cell-free transcription system. Template-commitment studies show that eucaryal TBP is stably sequestered at the archaeal promoter and that this interaction is further stabilized in combination with aTFA. Binding studies revealed that recognition of an archaeal promoter by TBP involves specific binding to the TATA box. These findings demonstrate a common function of TBP and aTFB and imply a common evolutionary origin of eucaryal and archaeal transcriptional machinery.

Base Sequence↗
PubMed1996

Two transcription factors related with the eucaryal transcription factors TATA-binding protein and transcription factor IIB direct promoter recognition by an archaeal RNA polymerase.

We reported previously that cell-free transcription in the Archaea Methanococcus and Pyrococcus depends upon two archaeal transcription factors, archaeal transcription factor A (aTFA) and archaeal transcription factor B (aTFB). In the genome of Pyrococcus genes encoding putative homologues of eucaryal transcription factors TATA-binding protein (TBP) and TFIIB have been detected. Here, we report that Escherichia coli synthesized Pyrococcus homologues of TBP and TFIIB are able to replace endogenous aTFB and aTFA in cell-free transcription reactions. Antibodies raised against archaeal TBP and TFIIB bind to polypeptides of identical molecular mass in the aTFB and aTFA fraction. These data identify aTFB as archaeal TBP and aTFA as the archaeal homologue of TFIIB. At the Pyrococcus glutamate dehydrogenase (gdh) promoter these two bacterially produced transcription factors and endogenous RNA polymerase are sufficient to direct accurate and active initiation of transcription. DNase I protection experiments revealed Pyrococcus-TBP producing a characteristic footprint between position -20 and -34 centered around the TATA box of gdh promoter. Pyrococcus-TFIIB did not bind to the TATA box but bound cooperatively with Pyrococcus-TBP generating an extended DNase I footprinting pattern ranging from position -19 to -42. These data suggest that the Pyrococcus homologue of TFIIB associates with the TBP-promoter binary complex as its eucaryal counterpart, but in contrast to eucaryal TFIIB, it causes an extension of the protection to the region upstream of the TATA box.

Archaea↗
PubMed2001

Identification of a conserved archaeal RNA polymerase subunit contacted by the basal transcription factor TFB.

Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promoters in vitro. These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB. Thus, the archaeal and eucaryal transcription machineries are fundamentally related. In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter. However the subunit(s) directly contacted by these factors has not been identified. Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK. Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (omega-subunit).

Amino Acid Sequence↗
PubMed2001

Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales.

Post-transcriptional modifications in archaeal RNA are known to be phylogenetically distinct but relatively little is known of tRNA from the Methanococci, a lineage of methanogenic marine euryarchaea that grow over an unusually broad temperature range. Transfer RNAs from Methanococcus vannielii, Methanococcus maripaludis, the thermophile Methanococcus thermolithotrophicus, and hyperthermophiles Methanococcus jannaschii and Methanococcus igneus were studied to determine whether modification patterns reflect the close phylogenetic relationships inferred from small ribosomal subunit RNA sequences, and to examine modification differences associated with temperature of growth. Twenty-four modified nucleosides were characterized, including the complex tricyclic nucleoside wyosine characteristic of position 37 in tRNA(Phe) and known previously only in eukarya, plus two new wye family members of presently unknown structure. The hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, reported previously only in bacterial tRNA at the first position of the anticodon, was identified by liquid chromatography-electrospray ionization mass spectrometry in four of the five organisms. The ribose-methylated nucleosides, 2'-O-methyladenosine, N(2),2'-O-dimethylguanosine and N(2),N(2),2'-O-trimethylguanosine, were found only in hyperthermophile tRNA, consistent with their proposed roles in thermal stabilization of tRNA.

Chromatography, High Pressure Liquid↗
PubMed2004

In vitro selection of an archaeal RNase P RNA mimics natural variation.

Archaeal and bacterial RNase P RNAs are similar in sequence and secondary structure, but in the absence of protein, the archaeal RNAs are much less active and require extreme ionic conditions for activity. To assess how readily the activity of the archaeal RNA alone could be improved by small changes in sequence, in vitro selection was used to generate variants of a Methanobacterium formicicum RNase P RNA: Bacillus subtilus pre-tRNA(Asp) self-cleaving conjugate RNA. Functional variants were generated with a spectrum of mutations that were predominately consistent with natural variation in this RNA. Variants generated from the selection had cleavage rates comparable to that of wild type; variants with improved cleavage rates or lower ionic requirements were not obtained. This suggests that the RNase P RNAs of Bacteria and Archaea are globally optimized and the basis for the large biochemical differences between these two types of RNase P RNA is distributed in the molecule.

Archaea↗
PubMed1996

Archaeal transcription factors and their role in transcription initiation.

Archaeal RNA polymerases show a weak ability in vitro to bind to promoter DNA and/or to initiate transcription with low activity independent of upstream regulatory DNA sequences. Active transcription in vitro and in vivo, however, depends strictly on a TATA box resembling the TATA box of eucaryal polII promoters. This TATA box is recognized by a polypeptide related to eucaryal TATA-binding protein (TBP) that was formerly designated aTFB. Template competition studies showed that this archaeal TATA-binding protein (aTBP) is stably sequestered at the promoter by interaction with the second archaeal transcription factor, aTFA, which is related to eucaryal transcription factor IIB (TFIIB). The association of archaeal TFIIB (aTFIIB) with the aTBP-promoter complex leads to template commitment, indicating that aTFIIB recruits archaeal RNA polymerase to the preinitiation complex. These analyses suggest the following order for assembly of transcription factors on the archaeal promoter: aTBP, aTFIIB, RNA polymerase, and provide evidence for a common molecular mechanism of transcription initiation by eucaryal RNA polymerase II and archaeal RNA polymerases. The sequence of the genes encoding aTBP and aTFIIB (TFB) showed all the characteristics conserved in their eucaryal counterparts. The degree of sequence similarity between archaeal and eucaryal transcription factors is between 27 to 35% for TFIIB and between 36 to 41% for TBP. The findings discussed here indicate that TBP and TFIIB perform analogous functions in Archaea and Eucarya and show that four essential components of archaeal and eucaryal transcriptional machineries. RNA polymerase, TATA box, TBP and TFIIB are homologous.

Archaea↗
PubMed2003

Two reactions of Haloferax volcanii RNA splicing enzymes: joining of exons and circularization of introns.

Archaeal RNA splicing involves at least two protein enzymes, a specific endonuclease and a specific ligase. The endonuclease recognizes and cleaves within a characteristic bulge-helix-bulge (BHB) structure formed by pairing of the regions near the two exon-intron junctions, producing 2',3'-cyclic phosphate and 5'-hydroxyl termini. The ligase joins the exons and converts the cyclic phosphate into junction phosphate. The ligated product contains a seven-base hairpin loop, in which the splice junction is in between the two 3' terminal residues of the loop. Archaeal splicing endonucleases are also involved in rRNA processing, cutting within the BHB structures formed by pairing of the 5' and 3' flanking regions of the rRNAs. Large free introns derived from pre-rRNAs have been observed as stable and abundant circular RNAs in certain Crenarchaeota, a kingdom in the domain Archaea. In the present study, we show that the cells of Haloferax volcanii, a Euryarchaeote, contain circular RNAs formed by 3',5'-phosphodiester linkage between the two termini of the introns derived from their pre-tRNAs. H. volcanii ligase, in vitro, can also circularize both endonuclease-cleaved introns, and non-endonuclease-produced substrates. Exon joining and intron circularization are mechanistically similar ligation reactions that can occur independently. The size of the ligated hairpin loop and position of the splice junction within this loop can be changed in in vitro ligation reactions. Overall, archaeal RNA splicing seems to involve two sets of two symmetric transesterification reactions each.

Exons↗
PubMed1993

Structure of the archaeal transfer RNA nucleoside G*-15 (2-amino-4,7-dihydro- 4-oxo-7-beta-D-ribofuranosyl-1H-pyrrolo[2,3-d]pyrimidine-5-carboximi dam ide (archaeosine)).

A number of post-transcriptional modifications in tRNA are phylogenetically characteristic of the bacterial, eukaryal, or archaeal domains, both with respect to sequence location and molecular structure at the nucleoside level. One of the most distinct such modifications is nucleoside G*, located in archaeal tRNA at position 15, which in bacterial and eukaryal tRNAs is a conserved site involved in maintenance of the dihydrouridine loop-T-loop tertiary interactions. G* occurs widely in nearly every branch of the archaeal phylogenetic domain, in contrast to its absence in all reported bacterial and eukaryal tRNA sequences. The structure of G*-15 is 2-amino-4,7-dihydro-4-oxo-7-beta-D-ribofuranosyl-1H- pyrrolo[2,3-d]pyrimidine-5-carboximidamide (7-formamidino-7-deazaguanosine), which is a non-purine, non-pyrimidine ribonucleoside; its structure thus reflects extensive modification beyond the guanine-15 specified by corresponding gene sequences. The structure was established by mass spectrometry, and in particular from collision-induced dissociation mass spectra of derivatives formed by microscale permethylation, and is confirmed by chemical synthesis.

Chromatography, High Pressure Liquid↗
PubMed2001

Similar subunit architecture of archaeal and eukaryal RNA polymerases.

Protein interactions among RNA polymerase small subunits from the archaeon Methanococcus jannaschii were investigated using affinity pulldown assays in pairwise and higher-order combinations. In the most extensive study of archaeal RNA polymerase subunit interactions to date, including 37 pairs of proteins, 10 ternary combinations, and three quaternary combinations, we found evidence for pairwise interactions of subunit D with subunits L and N, and a ternary complex of subunits D, L and N. No other small subunit interactions occurred. These results are consistent with interactions observed in a crystal structure of eukaryotic RNA polymerase II and support a common archaeal/eukaryal RNA polymerase architecture. We further propose that subunit E" is not an integral member of archaeal RNA polymerases. Finally, we discuss the relative accuracy of the various methods that have been used to predict protein-protein interactions in RNA polymerase.

Blotting, Western↗
PubMed2005

Direct modulation of RNA polymerase core functions by basal transcription factors.

Archaeal RNA polymerases (RNAPs) are recruited to promoters through the joint action of three basal transcription factors: TATA-binding protein, TFB (archaeal homolog of TFIIB), and TFE (archaeal homolog of TFIIE). Our results demonstrate several new insights into the mechanisms of TFB and TFE during the transcription cycle. (i) The N-terminal Zn ribbon of TFB displays a surprising degree of redundancy for the recruitment of RNAP during transcription initiation in the archaeal system. (ii) The B-finger domain of TFB participates in transcription initiation events by stimulating abortive and productive transcription in a recruitment-independent function. TFB thus combines physical recruitment of the RNAP with an active role in influencing the catalytic properties of RNAP during transcription initiation. (iii) TFB mutations are complemented by TFE, thereby demonstrating that both factors act synergistically during transcription initiation. (iv) An additional function of TFE is to dynamically alter the nucleic acid-binding properties of RNAP by stabilizing the initiation complex and destabilizing elongation complexes.

Amino Acid Sequence↗
PubMed2004

Transcriptional fidelity and proofreading in Archaea and implications for the mechanism of TFS-induced RNA cleavage.

We have addressed the question whether TFS, a protein that stimulates the intrinsic cleavage activity of the archaeal RNA polymerase, is able to improve the fidelity of transcription in Methanococcus. Using non-specific transcription experiments, we could demonstrate that misincorporation of non-templated nucleotides is reduced in the presence of TFS. A more detailed analysis revealed that elongation complexes containing a misincorporated nucleotide were arrested, but could be reactivated by TFS. RNase as well as exonuclease III footprinting experiments demonstrated that this arrest was not combined with extended backtracking. Analysis of paused elongation complexes demonstrated that TFS is able to induce a cleavage resynthesis cycle in such complexes, which resulted in the accumulation of dinucleotides corresponding to the last two nucleotides of the transcript. Further analysis of cleavage products revealed that, even under conditions that strongly promote misincorporation, still 50% of the released dinucleotides were correctly incorporated. Therefore, we assume that pausing of elongation complexes is an important determinant of TFS-induced RNA cleavage from the 3' end. As the incorporation rate of wrong nucleotides is about 700-fold reduced, it is possible that this delay also provides an appropriate time window for cleavage induction in order to maintain transcriptional fidelity by preventing misincorporation.

Archaea↗