| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700, USA
Correspondence
Julie A. Maupin-Furlow
jmaupin{at}ufl.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
1, β and 2, as well as panA and panB, encoding the PanA and PanB proteasome-activating nucleotidase proteins, respectively. All five of these genes are dispersed throughout the H. volcanii genome. For each proteasomal gene, a distinct transcript was detected by Northern blotting that was similar in size to the respective coding region. For both psmA and psmC, an additional transcript was detected that was 1.34 and 0.85 kb greater, respectively, than the coding region. Further analysis by Northern blotting and RT-PCR revealed that psmA was co-transcribed with genes encoding a Pop5 homologue of the RNase P endoRNase as well as an S-adenosylmethionine (SAM)-dependent methyltransferase. Likewise, psmC was co-transcribed with a downstream gene encoding a molybdenum cofactor sulfurase C-terminal (MOSC) domain protein. Additional proteasomal and neighbouring gene-specific transcriptional linkages were detected by RT-PCR. These results provide the first evidence that proteasome and tRNA modification genes are co-transcribed, reveal that a number of additional enzymes including those predicted to facilitate metal–sulfur cluster assembly are co-regulated with proteasomes at the transcriptional level, and provide further insight into proteasome gene transcription in archaea.
Supplementary data are available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). These enzymes form nanocompartments within the cell that selectively degrade specific proteins into oligopeptides that are hydrolysed to free amino acids by downstream peptidases. The 20S core, responsible for the proteolytic activity of proteasomes, is a cylindrical bundle of four stacked heptameric rings composed of related - and β-type subunits in an 7 : β7 : β7 : 7 configuration. Gated openings on each end of the core (formed by -type subunits) limit substrate access to the central proteolytic chamber (formed by β-type subunits). Members of the ATPases associated with various cellular activities (AAA) family, including the related regulatory particle ATPases (Rpts) of eucaryal 26S proteasomes and proteasome-activating nucleotidases (PANs) of archaea, associate with 20S proteasomes and stimulate the energy-dependent degradation of proteins (Smith et al., 2006).
The halophilic archaeon Haloferax volcanii synthesizes at least five proteasomal proteins, including two PAN proteins (PanA and PanB) and three 20S proteasomal subunits (1, β and 2) (Wilson et al., 1999; Reuter et al., 2004). The latter form at least two active 20S proteasome subtypes (1β and 12β) (Kaczowka & Maupin-Furlow, 2003). As is common among archaea, the genes encoding these various proteasomal proteins are dispersed throughout the genome. Although scattered, comparative genomics reveals that archaeal proteasome genes reside in what appear to be evolutionarily conserved superoperons that include non-proteasomal genes (Maupin-Furlow et al., 2000; Koonin et al., 2001).
Little is known regarding the transcripts generated from archaeal proteasome genes and the organization of these operons. In silico analyses of archaeal genome sequences (Maupin-Furlow et al., 2000; Koonin et al., 2001) reveal a high conservation of gene order between proteasomal genes and their neighbours, suggesting that these genes are linked at the transcriptional level. Northern blotting and primer-extension analysis of the 20S proteasomal genes (psmA and psmB encoding and β subunits) of the methanogenic archaeon Methanosarcina thermophila reveal single gene transcripts (Maupin-Furlow & Ferry, 1995). However, a TATA-like promoter element is only detected upstream of the M. thermophila psmB (vs psmA), thus suggesting that the psmA-specific transcript is modified by post-transcriptional RNA cleavages in this methanogen.
Regarding H. volcanii, our work has shown that transcripts specific for the genes encoding the five proteasomal proteins (PanA, PanB, and 20S proteasome 1, β and 2) are present in exponential phase, and the abundance of these transcripts increases in parallel as cells enter stationary phase (Reuter et al., 2004). Although the levels of PanA, 1 and β proteins remain relatively unchanged during this transition, the levels of PanB and 2 proteins increase severalfold, similarly to their encoding transcripts (Reuter et al., 2004). Thus, the levels of proteasome proteins appear to be regulated by transcriptional and post-transcriptional mechanisms.
To further understand the transcription of the H. volcanii proteasomal genes and the relationship of this to proteasome regulation and operon organization, proteasome-specific mRNAs were analysed by Northern blotting, RT-PCR and primer extension. This analysis is believed to provide the first evidence that ‘non-proteasomal’ genes [including RNase P, molybdenum cofactor sulfurase C-terminal (MOSC) domain and S-adenosylmethionine (SAM)-dependent methyltransferase homologues] are co-transcribed with genes encoding known proteasome proteins. This study also identified the 5' ends of proteasomal and neighbouring gene-specific transcripts, the results of which suggest that transcripts from the proteasomal regions of the H. volcanii genome undergo 5'-end post-transcriptional RNA cleavages.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Strains, media, plasmids and nucleic acid electrophoresis.
Bacterial and archaeal strains, oligonucleotide primers, template DNA and plasmids are summarized in Supplementary Tables S1–S4. Escherichia coli DH5 (New England BioLabs) was grown in Luria–Bertani (LB) medium (37 °C, 200 r.p.m.). H. volcanii DS2 (Mullakhanbhai & Larsen, 1975) was grown in ATCC 974 complex medium (42 °C, 200 r.p.m.). Media were supplemented with 100 mg ampicillin l–1 or 50 mg kanamycin l–1, as needed. Plasmids were purified using the Qiagen Prep Spin kit. DNA and RNA were separated by agarose gel electrophoresis (35 min, 5.6 V cm–1, 20 °C) using 0.8 and 2 % (w/v) agarose gels in 1x Tris/acetate/EDTA buffer, and detected by ethidium bromide according to standard procedures (Ausubel et al., 1987).
RNA purification.
Total RNA was isolated from H. volcanii DS2 (exponential phase; OD600 0.7) according to Nieuwlandt et al. (1995). RNA was treated with amplification grade DNase I according to the supplier's recommendations (Sigma-Aldrich), with the following modification: 3 U enzyme was added per microgram RNA and the mixture was incubated for 15 min at 37 °C. The integrity of RNA was determined by agarose gel electrophoresis. RNA concentration was determined by A260 using a Bio-Rad SmartSpec 3000 instrument.
Northern blot analysis.
Total RNA (12 µg per lane) was denatured and fractionated by electrophoresis (14 h, 20 V) using formaldehyde/0.8 % agarose gels in 1x MOPS buffer (20 mM MOPS, pH 7.0, 5 mM sodium acetate, 1 mM EDTA) using standard procedures (Ausubel et al., 1987). RNA molecular mass standards (0.24–9.5 kb RNA Ladder, Invitrogen Life Technologies) were included in the gels. RNA was transferred from gels to BrightStar-Plus membranes (Ambion) by downward capillary action for 3 h using 10 mM NaOH and 5x saline sodium citrate (SSC) (where 20x SSC is 3 M NaCl, 0.3 M sodium citrate, pH 7.0). RNA was cross-linked to membranes using a UV Stratalinker 2400 (Stratagene) and was hybridized to gene-specific probes or stained with 0.03 % (w/v) methylene blue in 0.3 M sodium acetate, pH 5.2. The probes were complementary nucleotide fragments of target gene-coding regions generated by linear amplification with digoxigenin-11-dUTP (Roche) and Taq DNA polymerase (New England Biolabs), according to the recommendations of the suppliers. The templates and primers used for amplification are summarized in Supplementary Table S2. For hybridization, membranes with cross-linked RNA samples were equilibrated in high SDS buffer (5x SSC, 2 %, w/v, blocking reagent, 0.1 %, w/v, N-lauroylsarcosine, 0.2 %, w/v, SDS, 50 %, w/v, formamide) (2 h, 55 °C), followed by incubation with 25 ng labelled probe per millilitre of high SDS buffer (16 h, 55 °C). Membranes were washed with 2x SSC supplemented with 0.1 % (w/v) SDS (two times 5 min, 25 °C) and 0.5x SSC supplemented with 0.1 % SDS (w/v) (two times 15 min, 55 °C). Hybridization products were detected by colorimetric and chemiluminescent (CSPD*) digoxigenin immunoassay according to the supplier's recommendations (Roche).
Primer-extension analysis.
The 5' ends of proteasomal operon-specific transcripts were mapped by primer extension. Total RNA (20 µg) was suspended in 5.5 µl nucleotide mix (7.5 µM dGTP, 7.5 µM dTTP and 7.5 µM dCTP), and 1 µl of 2 µM primer (listed in Supplementary Table S3) was added. Sample that was boiled (2 min), cooled (room temperature, 5 min) and equilibrated (42 °C, 1 min) was mixed with 2 µl 5x avian myeloblastosis virus reverse transcriptase (AMV RT) buffer (Promega) and incubated at 40 °C (10 min). AMV RT (11.5 U) and 1 µl [32P]dATP (3000 Ci mmol–1; 111 TBq mmol–1; Perkin Elmer) were added to the RNA, and the sample was incubated at 30 min for 40 °C. Freshly made quench solution (3 µl) (7.5 mM each of dATP, dCTP, dGTP and dTTP) was added and incubated for 30 min at 40 °C to complete the replication. Stop mix (13 µl) was then added, which was composed of 95 % (v/v) formamide, 20 mM EDTA, pH 8.0, 0.05 % (w/v) bromophenol blue and 0.05 % (w/v) xylene cyanol FF. For standards, DNA sequencing reactions using the same primers as those in the primer-extension reactions were performed with the plasmid DNA templates listed in Supplementary Table S3 and a Sequenase 7-deaza-dGTP Sequencing kit according to the supplier's recommendations (US Biochemicals). DNA sequencing and primer-extension reactions were separated by electrophoresis (3 h at 1800 V, 50 °C) using a high-resolution denaturing 6 % (w/v) polyacrylamide gel (Bio-Rad). Reaction products were visualized by autoradiography after exposure of Kodak RX Blue film to the dried polyacrylamide gels for 16–48 h at 20 °C.
RT-PCR.
RT-PCR was performed using H. volcanii total RNA (0.5 µg) as template, appropriate primers (Supplementary Table S3), OneStep RT-PCR reaction mix (Qiagen) and an iCycler (Bio-Rad). OneStep RT-PCR included Q-solution, Omniscript and Sensiscript reverse transcriptases, and HotStart Taq DNA polymerase. After cDNA synthesis (50 °C, 30 min), reactions were preheated to 95 °C (15 min), followed by 30 amplification cycles consisting of denaturation (94 °C, 30 s), annealing (30 s, temperatures listed in Supplementary Table S3) and elongation (72 °C, 1 min). Final extension was performed at 72 °C (10 min). For each primer pair, negative and positive controls were included to exclude genomic DNA contamination and confirm primer pair function, respectively. Controls were identical to the RT-PCR reactions, with the following exceptions: sample was maintained on ice during the reverse-transcription step for the negative control, and H. volcanii genomic DNA (10–25 ng) (Ng et al., 1995) was used as a template for the positive control.
RNA fold prediction.
Secondary structure and 
G values for RNA were estimated using mFold (Integrated DNA Technologies) using 50 maximum foldings, linear RNA, and 50 % suboptimality. Invariable parameters were 25 °C and 1 M sodium concentration.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
1), psmB (β), psmC (2), panA (PanA) and panB (PanB)] are dispersed throughout its 2.84 Mb chromosome (Wilson et al., 1999; Reuter et al., 2004). In this study, transcripts specific for each proteasomal gene were analysed by Northern blotting, primer extension and RT-PCR, using total RNA prepared from exponential-phase cells (see Supplementary Tables S1–S4 and Methods for details). Comparative genomics was also performed to understand how these transcripts correlate with gene neighbourhoods conserved within haloarchaea (i.e. H. volcanii, Haloquandratum walsbyi, Halobacterium sp. NRC-1, Haloarcula marismortui and Natronomonas pharaonis).
psmA (1) region
Northern blot analysis using a cDNA probe specific for psmA revealed two distinct transcripts of 1.0 and 2.1 kb, with the smaller transcript more abundant than the larger transcript (Fig. 1). Both transcripts were greater in size than the 0.76 kb coding capacity of psmA, thus suggesting that they may also be specific for gene neighbours. To investigate this, the genome sequence of H. volcanii (05/26/06 assembly; http://archaea.ucsc.edu/) was used to estimate the size of genes adjacent to psmA. The 234 bp region immediately downstream of psmA contained highly repetitive DNA sequence elements, lacked apparent coding capacity for protein, and preceded the termination codon of cinR (a gene encoded on the DNA strand complementary to psmA) (Fig. 2). The region upstream of psmA included rnpA, sam and rnpB genes in the same orientation as psmA, and respectively encoding RNase P Rpp30, SAM-dependent methyltransferase and RNase P Pop5 protein homologues. This rnpA-sam-rnpB-psmA region spanned a total of 2559 bp, with the coding sequences of rnpA and sam overlapping by 17 bp and the intergenic regions of sam-rnpB and rnpB-psmA at 9 and 6 bp, respectively. The 17 bp overlap of rnpA and sam suggested that these two genes were co-transcribed; however, neither of the psmA-specific transcripts detected by Northern blotting was large enough to accommodate rnpA to psmA. Instead, the larger (2.1 kb) transcript was similar in size to the 1.9 kb sam-rnpB-psmA coding sequence plus the 0.23 kb downstream region. The 1.0 kb size of the smaller transcript, in contrast, was not large enough to accommodate the 1.2 kb required to encode the complete upstream rnpB gene in addition to psmA. This smaller transcript was similar in size to psmA (0.76 kb) plus the 0.23 kb downstream region.
|
|
). A single transcript of 2.1 kb was detected with the sam- and rnpB-specific probes that was comparable in size to the larger transcript observed with the psmA-specific probe (Fig. 1). No signal was detected with the rnpA-specific probe (data not shown). Although the results do not clarify whether rnpA is co-transcribed with its gene neighbours, they do reveal that sam-rnpB-psmA are co-transcribed on a 2.1 kb transcript. Interestingly, a high degree of conservation was observed among haloarchaea and other archaea in which psmA genes were in the same orientation and linked with upstream rnpA and/or rnpB genes (Fig. 2) (Supplementary Fig. S1 for details). Similar to H. volcanii, SAM-dependent methyltransferase genes were also arranged between rnpA and rnpB genes in the Halobacterium sp. NRC-1 and Haloquandratum walsbyi genomes. Thus, the transcriptional linkage of psmA, rnpA, rnpB and sam genes is likely to be more widespread than in H. volcanii alone.
In addition to Northern blotting, RT-PCR was performed to analyse the transcriptional continuity of the genes within the psmA region (Fig. 2). Appropriate controls were included to confirm that each set of primer pairs was functional and to rule out the possibility that the RT-PCR product was due to genomic DNA contamination of the RNA samples (see Methods). RT-PCR products were analysed by agarose gel electrophoresis and DNA sequencing. Adjacent genes were characterized as either co-transcribed or not co-transcribed, based on the respective presence or absence of a single RT-PCR product homologous in size and sequence to that expected based on primer pair design. As for Northern blotting, transcriptional continuity between sam-rnpB-psmA was detected by RT-PCR (Fig. 2). Pairwise linkages were also detected for sam-rnpA, rnpA-ORF00853 and ORF00853-ORF00852, where ORF00852 and ORF00853 encode a putative M20/M25/M40-type peptidase/hydrolase and a conserved membrane protein, respectively. Together, these six ORFs (ORF00852 to psmA) span a total of 4.4 kb of coding sequence. It is unclear whether a large transcript is synthesized within the psmA region that spans all six of these ORFs or whether multiple promoters are used to generate smaller transcripts which harbour these gene pairs. If large mRNA species are generated, either they are not detected by the Northern blot methods of this study and/or they are cleaved post-translationally by RNases to generate smaller transcripts which harbour the coding capacity for a subset of these gene pairs. In addition to the linkages described above, ORF00850 and ORF00851 (lysA and dapF homologues) were co-transcribed but not linked to the ORF00852 to psmA region based on RT-PCR analysis.
The 5' ends of selected transcripts within the psmA region were mapped by primer extension. Distinct products were identified 142 and 141 bp upstream of the rnpA and psmA start codons, respectively (Fig. 2). The 5' ends of these transcripts began with either a C or a G, and thus were consistent with previously characterized haloarchaeal transcripts. In contrast, the DNA sequences 19–32 bases upstream of these transcript start sites (Figs 2 and 3) were GC rich (64 % G+C) and not related to the haloarchaeal TATA-like promoter consensus sequence described by Danner & Soppa (1996) (RGTWWWWRACYGSY, where R=A or G, Y=C or T, W=A or T, S=G or C). It is interesting to note that highly identical sequences were identified which were 38–20 bases upstream of the psmA- and psmB (β)-specific transcript start sites (Fig. 3) (CGSKGMCRRYGCGYGYCTG, where S=C or G, K=T or G, M=C or A, R=G or A, Y=C or T) (see discussion of psmB region, below). In addition, TATA-like (BoxA) sequences were identified 310 and 139 bp upstream of the start codon of rnpA and psmA, respectively (Fig. 2). While the Box A sequence upstream of psmA (b) is within the rnpB coding sequence, it overlaps (and thus cannot account for) the psmA-specific transcript start site. The second Box A sequence (a), in contrast, is positioned 168 bp upstream of the rnpA-specific transcript start site, thus opening the possibility that transcription initiates from this promoter-like element and that the resulting transcripts are cleaved at the 5' end to generate rnpA- and/or psmA-specific transcripts.
|
2) region). Of these, the smaller transcript was comparable in size to the 0.75 kb coding capacity of psmC, and the larger was consistent with the 1637 bp distance spanning the start codon of psmC to the stop codon of mosC (Fig. 4). In further support of the psmC-mosC transcriptional linkage, the stop codon of psmC was found to immediately precede the start codon of mosC. In contrast, ORF02058-psmC and ORF02059-ORF02058-psmC span 1088 and 2084 bp, with intergenic regions of 75 and 159 bp upstream of ORF02058 and psmC, respectively. RT-PCR analysis of the psmC region (Fig. 4) revealed transcriptional continuity between psmC and mosC, consistent with these Northern blot results. In addition, RT-PCR analysis demonstrated that the psmC-mosC gene pair was also linked at the transcriptional level to additional upstream and downstream ORFs (02059, 02058, 02055 and 02054) that encode conserved or hypothetical proteins (Fig. 4). The transcriptional linkage of this ORF02059 to ORF02054 region, which spans 4235 bp, was based on the RT-PCR products generated between ORF02059 and ORF02058, ORF02058 and psmC, psmC and mosC, mosC and ORF02055, and ORF02055 and ORF02054. The linkage of the last two gene pairs, however, is based on relatively weak RT-PCR products, suggesting that the levels of these transcripts are limited under the conditions examined in this study.
|
). Both transcripts began with a G and had relatively GC-rich sequences 19–32 bases upstream of their start sites. A Box A sequence (a) preceded by repeats of CTCGTGA was identified 426 bp upstream of the ORF02058-specific transcript start site. Likewise, a Box A sequence (b) was identified 251 bp upstream of the psmC-specific transcript start site. The location of both of these Box A elements supports the suggestion that ORF02058- and psmC-specific transcripts undergo processing after expression from Box A (a) and (a or b), respectively. Although the primary (unprocessed) transcript generated from Box A (b) would include the 4 bases upstream of ORF02058, it would require ORF02058 to be translated using an internal ribosome-binding site. Likewise, the third Box A-like sequence 29 bp upstream of psmC (c) would generate a leaderless transcript devoid of the psmC-Shine–Dalgarno site. The examples of this type of translation initiation occurring in archaea, however, are limited (Srinivasan et al., 2006).
Comparative genomics of the psmC region was performed among archaea which encode a second -type 20S proteasomal subunit (H. volcanii, Haloquandratum walsbyi and Haloarcula marismortui) (see Supplementary Fig. S2 for details). Of these, Haloarcula marismortui was unique, with a coding capacity for both 2 and β2 in a region that to date displays little or no gene conservation with other organisms. In contrast, the psmC regions of H. volcanii and Haloquandratum walsbyi were somewhat related. Although the genes immediately 5' and 3' of the H. volcanii psmC gene are not conserved, the gene neighbours further upstream are conserved with Haloquandratum walsbyi, including thyA (thymidylate synthase) and folA (dihydrofolate reductase).
psmB (β) region
Northern blot analysis with a psmB-specific cDNA probe detected a single 0.75 kb transcript which correlated in size to the 0.73 kb psmB coding region (Figs 1 and 5). Primer-extension analysis mapped the 5' end of the psmB-specific transcript to a G residue within the intergenic region and 49 bp upstream of the translation start site of psmB (Fig. 5). Although a Box A consensus was not identified 32–19 bases upstream of this transcript start site, the –38 to –20 region was 57 % identical to a sequence in a similar location upstream of the psmA-specific transcript start site (Figs 3 and 5). Based on the homology of these two regions, this GC-rich sequence may serve as an alternative promoter that coordinates the stoichiometric ratios of 1 and β proteins in 20S proteasome subtypes which are predominant during exponential-phase growth (Kaczowka & Maupin-Furlow, 2003). However, a number of Box A-like sequences (b, c, e) were also detected 305–446 bp upstream of the psmB-transcript start site (Fig. 5), thus opening the alternative possibility that a larger transcript is synthesized from one or more of these Box A promoters and cleaved at the 5' end to generate the psmB-specific transcript. A Box A consensus sequence located only 19 bp upstream of the psmB start codon was also identified (f) (Fig. 5); however, a leaderless psmB-specific transcript would be synthesized that would require internal translation initiation.
|
) that was not identified by Northern blotting. The orientation of this ORF in relation to psmB was highly conserved in the genomes of H. volcanii, Haloquandratum walsbyi and Haloarcula marismortui (Fig. 5) (see Supplementary Fig. S3 for details). In addition, two ORFs were conserved and typically in a divergent orientation with respect to the psmB genes of all haloarchaeal genomes to date. These ORFs included a conserved helix-turn-helix transcriptional regulator with a cystathionine β-synthase (CBS) domain proposed to bind the adenosyl portion of molecules such as SAM, ATP and AMP (Ignoul & Eggermont, 2005), as well as a DUF555 family member with a predicted central TM-spanning helix (Fig. 5; ORF00378 and ORF00377 for H. volcanii, respectively) (see Supplementary Fig. S3 for details).
panA region
A single panA-specific transcript of 1.4 kb, similar in size to the 1.22 kb panA-coding region, was detected by Northern blot analysis (Figs 1 and 6). In addition, a distinct primer-extension product was identified at a C residue located 181 bp upstream of the translation start site of panA (Fig. 6). Although the region 19–32 bases upstream of the transcript start site was relatively GC-rich, two sequences with similarity to the Box A consensus were identified within the arsR coding sequence 81–117 bp upstream of the transcript start site (a, b), suggesting that the panA-specific transcript is cleaved at the 5' end. Interestingly, two Box A-like sequences were also identified downstream of the primer-extension site that were within 110–129 bp of the panA start codon (d, f) and which overlapped complementary strand Box A-like sequences (c, e) predicted to mediate arsR transcription. While these do not account for the primer-extension product detected in this study, the consensus of these four Box A-like sequences is high, ranging from 64 to 86 % identity. Whether these promoter elements are negatively regulated during exponential-phase growth remains to be determined.
|
) that was not detected by Northern blotting. Comparison of haloarchaeal gene neighbourhoods revealed that this panA-pdg linkage was conserved in Haloquandratum walsbyi (Fig. 6) (see Supplementary Fig. S4 for details). In addition, the majority of haloarchaeal panA genes (including that of H. volcanii) were divergently orientated with arsR-, mre11- and rad50-like genes (respectively encoding homologues of ArsR transcriptional regulator, Mre11 DNA double-strand break repair, and Rad50 chromosome segregation proteins) (Fig. 6) (see Supplementary Fig. S4 for details). Thus, haloarchaeal panA genes may be transcriptionally regulated with pdg as well as other genes that encode DNA binding/modifying enzymes.
panB region
A single transcript of 1.35 kb was detected by Northern blotting with a panB-specific probe, which correlated in size to the 1.24 kb coding region of panB (Figs 1 and 7). This single gene transcript was also detected by RT-PCR, and its 5' end was mapped by primer extension (Fig. 7). Based on the latter analysis, a single distinct product was identified which mapped to a C residue within the intergenic region 221 bp upstream of the panB translation start site (Fig. 7). Unlike the other proteasomal genes, the region 19–32 bases upstream of this transcript start site was related to a Box A consensus with 57 % identity. Two additional Box A-like sequences were identified with similar identity; however, both were downstream of the transcript start site.
|
) (see Supplementary Fig. S5 for details). N-terminal protein acetyltransferase (rimI) and aconitate hydratase (citB) genes were divergently oriented from the panB genes of both of these haloarchaea. In addition, homologues of deaminase (dcd) and decarboxylase (pdaD) genes were in the same orientation and immediately surrounding these panB genes. A pdaD homologue was also detected downstream of the Halobacterium sp. NRC-1 panB. In H. volcanii, this pdaD gene homologue was linked to ORF03057 (a signal-transducing histidine kinase of COG0642) at the transcript level by RT-PCR (Fig. 7). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In contrast, RT-PCR is more sensitive and requires less manipulation of RNA, thus reducing the risk of mRNA degradation prior to analysis.
In addition to discovering transcriptional linkages in the proteasome-encoding regions of the genome, the 5' ends of proteasomal gene-specific transcripts were determined. With the exception of panB, relatively GC-rich sequences were located 19–32 bases upstream of the transcript start site that were not related to the Box A (TATA) consensus sequence common to many archaeal gene promoters (Bell & Jackson, 2001). It is possible that novel promoter elements drive transcription of these genes (most notably a conserved GC-rich sequence 20–38 bases upstream of the psmA and psmB transcript start sites). Haloarchaea are particularly complex in the number of transcription factor IIB (TFB) and TATA-binding protein (TBP) pairs which appear to recruit RNA polymerase to promoters, suggesting that more than one TATA-like consensus sequence is utilized in these organisms (Baliga et al., 2000; Facciotti et al., 2007). Alternatively, the transcripts mapped in this study may have arisen by posttranscriptional RNA cleavages at the 5' end. This latter possibility is supported by the finding of TATA-like (Box A) consensus sequences 81–426 bp upstream of the transcript start sites.
Although the neighbouring genes that were linked to proteasomal genes at the transcriptional level have yet to be characterized in H. volcanii, several of the deduced proteins are closely related to orthologous enzymes with known biochemical properties. These include rnpA and rnpB, which encode the Rpp30 and Pop5 protein subunits of RNaseP, an enzyme responsible for processing the 5' ends of precursors to tRNA as well as cleaving other stable RNAs, intergenic transcripts (Li & Altman, 2003) and riboswitches (Altman et al., 2005). Although the RNA component of the archaeal RNase P alone is capable of catalysing 5'-end tRNA maturation, substrate specificity (kcat/Km) is dramatically enhanced in the presence of Pop5 and Rpp30 (Tsai et al., 2006). In addition to RNase P proteins, the deduced protein of the H. volcanii pdg is closely related (E value 2x10–32) to the pyrimidine dimer glycosylase (UV endonuclease) of Micrococcus luteus (Piersen et al., 1995).
The function of remaining genes transcribed with the proteasomal genes and/or proteasomal gene neighbours is less clear, as no close homologues have been characterized. The 17 bp overlap of sam and rnpA suggests that both genes may be involved in tRNA modification. Consistent with this, the sam-deduced protein has the sequence motifs (I–III) common to protein, DNA, RNA and small-molecule SAM-dependent methyltransferases (Ingrosso et al., 1989) (Pfam08241; E value 2.1x10–14); however, the specificity of this group of enzymes is difficult to predict by bioinformatics alone. The mosC-encoded protein harbours an N-terminal β-barrel MOSC domain, including the highly conserved Cys predicted to accept sulfur (S0) from cysteine desulfurase (NifS-like) enzymes and deliver the abstracted sulfur to apoprotein targets during the assembly of metal–sulfur clusters (Anantharaman & Aravind, 2002). ORF02054 and ORF00852 respectively encode proteins related to metal-dependent hydrolases (cd01299 subgroup A; E value 4x10–44) and M20/M25/M40 peptidases (pfam01546; E value 1x10–28). Thus, these enzymes may be involved in peptide bond cleavage. The remaining ORFs (00853, 00376, 02059, 02058 and 02055) are conserved only with proteins of unknown function, including a subset predicted to be membrane-associated.
The reason for the assortment of putative RNA-modifying, apoprotein-maturation, DNA-repair and hydrolytic genes co-transcribed with the proteasomal genes remains to be determined. However, co-regulation of RNase P with its neighbours is not unusual, having also been observed in bacteria, in which rnpA (encoding C5 of RNase P) is linked at the transcriptional level to rmpH (encoding the small ribosomal protein L34) (Ellis & Brown, 2003). The transcriptional connections of RNase P to the proteasomes of H. volcanii are likely to serve multiple functions, including coordinating the balance of free amino acids generated by protein degradation (mediated by the proteasome and peptidases) with the free amino acids needed to charge tRNA after maturation by enzymes such as RNase P. Linking the transcription of proteasomal genes to those involved in apoprotein maturation (e.g. mosC) may enhance overall protein quality control. AAA+ proteins such as those associated with proteasomes (i.e. the PAN proteins) are important in the disaggregation and unfolding of proteins (Benaroudj & Goldberg, 2000), and thus may assist a ‘MosC-dependent’ metalloenzyme assembly pathway. Proteasomes also appear to be closely tied to the regulation of DNA repair (Sweder & Madura, 2002; Wang et al., 2005); the transcriptional linkage of panA to pdg may facilitate this type of regulation in H. volcanii.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: J. van der Oost
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Anantharaman, V. & Aravind, L. (2002). MOSC domains: ancient, predicted sulfur-carrier domains, present in diverse metal-sulfur cluster biosynthesis proteins including molybdenum cofactor sulfurases. FEMS Microbiol Lett 207, 55–61.[Medline]
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Seidman, J. G. & Struhl, K. (1987). Preparation and analysis of RNA. In Current Protocols in Molecular Biology, pp. 4.0.1–4.10.11. New York: Green Publishing Associates and Wiley-Interscience.
Baliga, N. S., Goo, Y. A., Ng, W. V., Hood, L., Daniels, C. J. & DasSarma, S. (2000). Is gene expression in Halobacterium NRC-1 regulated by multiple TBP and TFB transcription factors?. Mol Microbiol 36, 1184–1185.[CrossRef][Medline]
Bell, S. D. & Jackson, S. P. (2001). Mechanism and regulation of transcription in archaea. Curr Opin Microbiol 4, 208–213.[CrossRef][Medline]
Benaroudj, N. & Goldberg, A. L. (2000). PAN, the proteasome-activating nucleotidase from archaebacteria, is a protein-unfolding molecular chaperone. Nat Cell Biol 2, 833–839.[CrossRef][Medline]
Danner, S. & Soppa, J. (1996). Characterization of the distal promoter element of halobacteria in vivo using saturation mutagenesis and selection. Mol Microbiol 19, 1265–1276.[CrossRef][Medline]
Dvorak, Z., Pascussi, J. M. & Modriansky, M. (2003). Approaches to messenger RNA detection – comparison of methods. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 147, 131–135.[Medline]
Ellis, J. C. & Brown, J. W. (2003). Genes within genes within bacteria. Trends Biochem Sci 28, 521–523.[CrossRef][Medline]
Facciotti, M. T., Reiss, D. J., Pan, M., Kaur, A., Vuthoori, M., Bonneau, R., Shannon, P., Srivastava, A., Donohoe, S. M. & other authors (2007). General transcription factor specified global gene regulation in archaea. Proc Natl Acad Sci U S A 104, 4630–4635.
Ignoul, S. & Eggermont, J. (2005). CBS domains: structure, function, and pathology in human proteins. Am J Physiol Cell Physiol 289, C1369–C1378.
Ingrosso, D., Fowler, A. V., Bleibaum, J. & Clarke, S. (1989). Sequence of the D-aspartyl/L-isoaspartyl protein methyltransferase from human erythrocytes. Common sequence motifs for protein, DNA, RNA, and small molecule S-adenosylmethionine-dependent methyltransferases. J Biol Chem 264, 20131–20139.
Kaczowka, S. J. & Maupin-Furlow, J. A. (2003). Subunit topology of two 20S proteasomes from Haloferax volcanii. J Bacteriol 185, 165–174.
Koonin, E. V., Wolf, Y. I. & Aravind, L. (2001). Prediction of the archaeal exosome and its connections with the proteasome and the translation and transcription machineries by a comparative-genomic approach. Genome Res 11, 240–252.
Li, Y. & Altman, S. (2003). A specific endoribonuclease, RNase P, affects gene expression of polycistronic operon mRNAs. Proc Natl Acad Sci U S A 100, 13213–13218.
Maupin-Furlow, J. A. & Ferry, J. G. (1995). A proteasome from the methanogenic archaeon Methanosarcina thermophila. J Biol Chem 270, 28617–28622.
Maupin-Furlow, J. A., Wilson, H. L., Kaczowka, S. J. & Ou, M. S. (2000). Proteasomes in the archaea: from structure to function. Front Biosci 5, d837–d865.[CrossRef][Medline]
Maupin-Furlow, J. A., Humbard, M. A., Kirkland, P. A., Li, W., Reuter, C. J., Wright, A. J. & Zhou, G. (2006). Proteasomes from structure to function: perspectives from archaea. Curr Top Dev Biol 75, 125–169.[Medline]
Mullakhanbhai, M. F. & Larsen, H. (1975). Halobacterium volcanii spec. nov., a Dead Sea Halobacterium with a moderate salt requirement. Arch Microbiol 104, 207–214.[CrossRef][Medline]
Ng, W.-L., Yang, C.-F., Halladay, J. T., Arora, P. & DasSarma, S. (1995). Isolation of genomic and plasmid DNAs from Halobacterium halobium. In Archaea: a Laboratory Manual, pp. 179–184. New York: Cold Spring Harbor Laboratory Press.
Nieuwlandt, D. T., Palmer, J. R., Armbruster, D. W., Kuo, Y. P., Oda, W. & Daniels, C. J. (1995). A rapid procedure for the isolation of RNA from Haloferax volcanii. In Archaea: a Laboratory Manual, pp. 161–162. Plainview, NY: Cold Spring Harbor Laboratory Press.
Piersen, C. E., Prince, M. A., Augustine, M. L., Dodson, M. L. & Lloyd, R. S. (1995). Purification and cloning of Micrococcus luteus ultraviolet endonuclease, an N-glycosylase/abasic lyase that proceeds via an imino enzyme-DNA intermediate. J Biol Chem 270, 23475–23484.
Reuter, C. J., Kaczowka, S. J. & Maupin-Furlow, J. A. (2004). Differential regulation of the PanA and PanB proteasome-activating nucleotidase and 20S proteasomal proteins of the haloarchaeon Haloferax volcanii. J Bacteriol 186, 7763–7772.
Smith, D. M., Benaroudj, N. & Goldberg, A. (2006). Proteasomes and their associated ATPases: a destructive combination. J Struct Biol 156, 72–83.[Medline]
Srinivasan, G., Krebs, M.P. & RajBhandary, U.L. (2006). Translation initiation with GUC codon in the archaeon Halobacterium salinarum: implications for translation of leaderless mRNA and strict correlation between translation initiation and presence of mRNA. Mol Microbiol 59, 1013–1024.[CrossRef][Medline]
Sweder, K. & Madura, K. (2002). Regulation of repair by the 26S proteasome. J Biomed Biotechnol 2, 94–105.[CrossRef][Medline]
Tsai, H. Y., Pulukkunat, D. K., Woznick, W. K. & Gopalan, V. (2006). Functional reconstitution and characterization of Pyrococcus furiosus RNase P. Proc Natl Acad Sci U S A 103, 16147–16152.
Wang, Q. E., Wani, M. A., Chen, J., Zhu, Q., Wani, G., El Mahdy, M. A. & Wani, A. A. (2005). Cellular ubiquitination and proteasomal functions positively modulate mammalian nucleotide excision repair. Mol Carcinog 42, 53–64.[CrossRef][Medline]
Wilson, H. L., Aldrich, H. C. & Maupin-Furlow, J. A. (1999). Halophilic 20S proteasomes of the archaeon Haloferax volcanii: purification, characterization and gene sequence analysis. J Bacteriol 181, 5814–5824.
Received 19 March 2007;
revised 24 May 2007;
accepted 29 May 2007.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICR |
with kb) and 5' ends of transcripts mapped by primer extension (P1 and/or P2) are also indicated. Sequences with similarity to the TATA-like (Box A) promoter sequence described by Danner & Sopper (1996)
+1); regions 19–32 bp upstream of the transcript start site in boxes; ORF coding sequence with start and stop codons in bold type, putative Shine–Dalgarno (SD) sites in boxes; Box A promoter sequences in boxes with bases identical to the TATA consensus in bold italic type; stem–loop structures (
); and repetitive DNA sequences in bold italic type (not boxed).
