From the
Departments of Microbiology and
Immunology and ¶Medicine, University of Arizona
College of Medicine, Tucson, Arizona 85724-5049 and
Josephine Bay Paul Center for Comparative
Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole,
Massachusetts 02543-1015
Received for publication, March 31, 2003 , and in revised form, May 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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Gene organization and transcription in Giardia is unusual relative to more commonly studied eukaryotes. Most Giardia transcripts have a very short 5'-untranslated region (16 nucleotides) (6) and do not possess a 7-methyl guanosine cap (7). Because many of the intergenic regions are less than 100 base pairs in length (6) and can be as short as 810 bp,1 the very compact nature of the genome might prevent the general use of longer 5'-untranslated regions. Because of the potential impact on organization of promoter regions, these short intergenic elements may constrain the regulation of transcription. Eukaryotic RNA polymerase II (RNAP II)2 promoters are highly conserved and usually have 3 short sequences centered at 30 (TATA box), 75 (CAAT box), and 90 (GC box) bases upstream of the transcription start site (8). In contrast, the G. lamblia RNAP II promoters have AT-rich sequences at the transcription start site (consensus AATTAAAAA), 2035 nucleotides upstream of the start site (consensus CAAAAA(A/T)(T/C)AGA(G/T)TC(C/T)GAA), and a third hexamer 4070 bases upstream of the start site (consensus CAATTT) (9, 10). These differences in Giardia transcription suggest the possibility that the components of G. lamblia RNAP II that affect the transcription start site may differ from those of other eukaryotes.
The RNAP II holoenzyme in eukaryotes consists of 12 subunits, Rpb1 through Rpb12 (1116). Rpb1, Rpb2, Rpb3, Rpb4, Rpb7, Rpb9, and Rpb11 are unique to RNAP II, whereas Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12 are also components of the other two nuclear RNA polymerases (RNA polymerases I and III) (16). Rpb4 and Rpb9 are dispensable in Saccharomyces cerevisiae at optimal growth conditions, whereas the remaining subunits are essential for viability (17, 18).
The three nuclear RNA polymerases vary in their sensitivities to the cyclic
octapeptide -amanitin. RNAP I is resistant to amanitin, RNAP II is 50%
inhibited by 520 µg/ml, and RNAP III is 50% inhibited by 250
µg/ml amanitin. These differences in sensitivity are frequently exploited
to study the transcription of genes by different polymerases. For example, the
amanitin resistance of variant surface glycoprotein gene transcription in
African trypanosomes provided the basis for the proposal that these genes are
transcribed by RNAP I
(19).
However, there are organisms in which RNAP II transcription is naturally amanitin-resistant. RNAP II transcription in Trichomonas vaginalis is resistant to 250 µg/ml amanitin (20, 21), whereas Entamoeba histolytica transcription is resistant to 1 mg/ml amanitin (22). In the current manuscript, we describe the genes encoding G. lamblia RNAP II and demonstrate a potential molecular basis for amanitin-resistant transcription of protein-coding genes in G. lamblia.
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EXPERIMENTAL PROCEDURES |
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Identification of G. lamblia rpb GenesThe Giardia genome project (28) is now at 7.7-fold coverage with an estimated 99% coverage with at least a single pass. To identify the Rpb subunits of Giardia, we used the protein sequences of the Schizosaccharomyces pombe rpb genes as a query in a TBLASTN search against the G. lamblia project data base (www.mbl.edu/Giardia). We then analyzed the contigs containing the putative rpb genes. Because introns are rare in Giardia (one has been identified in the 2Fe2S ferredoxin gene (29)), we used the ORFs of the primary genomic sequences for subsequent analysis. We used the translation of each putative rpb ORF as a query in a BLASTP search against the nonredundant data base to confirm whether it was the ortholog of the given gene. When bidirectional sequences were not available, we used primer walking to obtain complete bidirectional sequences.
Using the S. pombe ortholog as a query, BLAST failed to identify the rpb7 gene in the genome data base, but a consensus of two conserved motifs from S. cerevisiae, Caenorhabditis elegans, Mus musculus, Rattus norvegicus, and Homo sapiens returned a putative rpb7 sequence with a TBLASTN score of 0.24.
rpb ProbesWe prepared probes for each rpb gene to determine genomic location and organization as well as transcript length. The ORFs of the three largest subunits, Rpb1, Rpb2, and Rpb3, were found entirely within the plasmid clones MJ3673, NJ2874, and KJ2040 (www.mbl.edu/Giardia); therefore, these plasmids were used as probes. In the case of rpb3, we used the plasmid clone KJ2040 for pulsed-field gel electrophoresis hybridizations and an antisense oligo (described below) to probe genomic restriction fragments and RNA blots. We used PCR products amplified from genomic DNA with the following primers as probes for the following rpb ORFs. The number indicated in the brackets refers to the size of the amplified product. TACCAACAGTCTCCGACG and GTCTACGGAACGGCAGAG for rpb5a (651), ATGGACTCACTTGCGCTG and AGAGATGTTTCCCCGCAC for rpb5b (586), GCCCTTGATCGTGGAAAG and GTAACTCGCTGAGCTTCC for rpb6 (283), CTTCCACATTGAGGCCAC and CCCCCCTATTGATGTTTC for rpb7 (382), ATCATCCCTGTTCGGTGC and TTACTTCTGCTCCGCCGC for rpb10 (241), CGGATCGGTACAAAACAC and GTGGGTGTGCATGATACT for rpb11 (356), and GGGAAAATCAGCCTGTCG and GATTAGGGTTTGTGTTGC for rpb12 (96). We used single-stranded antisense oligonucleotide probes for rpb3 (5'-GGGAGCCTTTCAAATTCATCGCAC) and rpb8 (5'-GCATAATCTAAGTCTGGGTAGGCG). Because of the small size of rpb12, we used reverse transcription-PCR with primers rpb12A (GGGAAAATCAGCCTGTCG) and rpb12B (GATTAGGGTTTGTGTTGC) to analyze the transcription of rpb12. The EZ rTth RNA PCR kit (Applied Biosystems, Foster City, CA) was used to amplify a 96-bp product within the rpb12 ORF. Procedures were followed as suggested by the manufacturer, except that a 50 °C annealing temperature was used instead of 60 °C. Sequences similar to rpb4 and rpb9 do not occur in the current coverage of the G. lamblia genome.
Identification of the G. lamblia Leucyl-tRNA GeneThe BLAST annotation of the Giardia genome sequences identified a putative leucyl-tRNA. The G. lamblia sequence was aligned with the C. elegans leucyl-tRNA sequence using LALIGN (30), using a +5/4 scoring matrix with gap and extension penalties of 12 and 4, respectively. An oligonucleotide with the sequence complementary to the identified gene (5'-GCCAGCTGTGGGGTTCGAACCCACGCGGTCTTGCAACCAATGGGACTTGAATCCATCGCCTTAACCACTCGG CCAAACTGGC) was used to probe northern blots as well as run-on transcripts.
Electrophoresis and Hybridization of Nucleic AcidsTotal
chromosomal DNA was prepared and separated by pulsed-field gel electrophoresis
using the ISR E11 isolate to identify chromosomal locations because
chromosomes 1 and 2 of this isolate can be readily distinguished
(26). Restriction enzyme
digestion and agarose gel electrophoresis of DNA were performed by standard
methods. DNA was transferred to a nylon membrane by alkaline transfer in 0.4
M NaOH. RNA was extracted using the RNeasy kit (Qiagen, Valencia,
CA), separated on a formaldehyde gel, and transferred to nylon in 10x
SSC (1x SSC = 0.15 M NaCl and 0.015 M sodium
citrate). Double-stranded DNA probes were labeled by random priming, and
unincorporated nucleotides were removed by Sephadex G-50 spun column
chromatography. Hybridization to DNA was performed in 5x SSC at 50
°C, and hybridization to RNA was performed in 5x SSC and 50%
formamide at 37 °C. Oligonucleotide probes were end-labeled with
[-32P]ATP and T4 polynucleotide kinase and purified using a
G-25 column. Hybridization was carried out in 6x SSC, and washing was
done in 1x SSC at 50 °C.
Nuclear Run-on AssaysG. lamblia nuclear run-on assays were performed as previously described for Trypanosoma brucei (31) and G. lamblia (32), with minor modifications. Trophozoite cultures were harvested in late log phase by centrifugation at 1200 x g and washed twice in phosphate-buffered saline. The pellets were re-suspended in Buffer A (150 mM sucrose, 20 mM KCl, 3 mM MgCl2, 20 mM HEPES, 1 mM dithiothreitol, 10 µg/ml leupeptin (Sigma-Aldrich) to a density of 6 x 108 cells/ml in 400-µl aliquots. Palmitoyl lysophosphatidylcholine (lysolecithin) was added to a concentration of 500 µg/ml and chilled on ice for 1 min. Two volumes of Buffer A at room temperature were added, and the trophozoites were then recovered by centrifugation as described before. The pellets were washed twice in Buffer A and resuspended in 100 µl of the run-on mixture (50 mM Tris-HCl, pH 7.9, 100 mM KCl, 5 mM MgCl2, 1 mM MnCl2, 2 mM dithiothreitol, 4 mM ATP, 2 mM GTP, 2 mM CTP, 100 µCi of [32P]UTP), 1 unit/µl RNasin (Promega, Madison, WI), 10 mM phosphocreatine, and 1.2 µg/µl creatine kinase). In the amanitin experiments, amanitin was added at concentrations of 0, 50, 250, or 1000 µg/ml. The reaction was carried out for 1 h at room temperature and stopped by adding DNase I (0.2 mg/ml) and proteinase K (25 mg/ml) followed by incubation at 55 °C for 30 min. Radiolabeled RNA was extracted from the cells using the DNA/RNA kit procedure for RNA extraction (Qiagen). DNA probes used for nuclear run-on assays were denatured and slot-blotted onto 0.45 µM Supercharge Nytran membranes (Schleicher and Schuell).
For the S. cerevisiae run-on assays, a log phase culture of S.
cerevisiae was harvested by centrifugation at 4600 x g.
The pellet (3 x 107 cells) was washed with TMN buffer (10
mM Tris HCl, pH 7.4, 100 mM NaCl, 5 mM
MgCl2) and resuspended in 1 ml of 0.5% Sarkosyl. The suspension was
placed on ice for 15 min, and the cells were recovered by centrifugation at
4000 x g for 1 min at 4 °C. The pellet was resuspended in
100 µl of run-on mixture (50 mM Tris-HCl pH 7.9, 100
mM KCl, 5 mM MgCl2, 1 mM
MnCl2, 2 mM dithiothreitol, 0.5 mM ATP, GTP,
and CTP, 100 µCi of [32P]UTP, 1 unit/µl RNasin, 10
mM phosphocreatine, and 1.2 µg/µl creatine kinase). In the
-amanitin control experiments,
-amanitin (50 µg/ml) was added
to the run-on mixture (33).
The reaction was carried out for 1 h at room temperature and stopped by adding
DNase I (0.2 mg/ml) and proteinase K (25 mg/ml) followed by incubation at 55
°C for 10 min. RNA was then extracted from the run-on mixture as for
G. lamblia and hybridized to the slot-blotted nucleic acid probes
(discussed below) in 6x SSC and 25% formamide at 37 °C. The blots
were washed in 1x SSC at 50 °C. In the RNase A control experiment,
the extracted RNA was incubated at 37 °C in the presence of 1 mg/ml RNaseA
for 1 h before hybridization.
Probes Used in Nuclear Run-on AssaysA plasmid probe for
ribosomal DNA in Giardia was obtained from Tom Edlind
(34). -Giardin
(35,
36) and vsp conserved
region (37) probes were
amplified from genomic DNA using the primers listed below. The vsp
conserved primers were based on the vspA6 (CRP170)
(38) sequence. In that region,
all published vsp sequences demonstrate greater than 90% nucleotide
identity; thus, all vsp transcripts should hybridize to the probe
(37).3
The probes for
-giardin (810 bp) (GenBankTM accession number GI
X85958
[GenBank]
) and the vsp conserved region (93 bp) (GenBankTM
accession number GI M83933
[GenBank]
) were obtained by means of PCR from genomic DNA
using the primer sets ATGTTCACCTCCACCCTTACG/GTGCTTTGTGACCATCGAGAG for
-giardin and GGTGCCATCGCGGGGATCTCC/CGCCTTCCCTCTACAG ATGAA for the vsp
conserved region.
Custom made oligo 60-mers,
GGCAAGCACGTCCCGCGCGCGGTCTTCGTTGACCTCGAGCCCACGGTCGTC GACGAGGTC and
GACCTCGTCGACGACCGTGGGCTCGAGGTCAACGAAGACCGCGCGCGGGACGTGCTTGCC, which are the
sense and antisense strands of the 2-tubulin gene, were used as single
strand DNA probes. Probes for the S. cerevisiae genes were amplified
from genomic DNA using the primers GGTTGATCCTGCCAGTAGTCATATG and
GACTTGCCCTCCAATTGTTCCTCG for 18 S rRNA (783-bp product)
(39),
ATATCACGGCCATGACGATATCCAG and GGCATCAAACATTTGCTGTG for
-tubulin (531-bp
product; GenBankTM accession number GI V01296
[GenBank]
), and
CGTTGTCGTTATCGGTCATGTCG and CGAATGGAACAGTCTTTGGGTTG for the
EF-1
gene (538 bp; GenBankTM accession number GI
X00779
[GenBank]
).
MEME AnalysisThe Rpb1 sequences of M. musculus
(GenBankTM U37500
[GenBank]
), H. sapiens (X63564
[GenBank]
), C. elegans
(T29959
[GenBank]
), Drosophila melanogaster (P04052
[GenBank]
), Arabidopsis
thaliana (CAB81489
[GenBank]
), Helobdella stagnalis (AAA50227
[GenBank]
), S.
pombe (AL121795
[GenBank]
), S. cerevisiae (X03128
[GenBank]
), Aspergillus
oryzae (AB017184
[GenBank]
), Plasmodium falciparum (NP_473294
[GenBank]
),
Leishmania major (AF009163
[GenBank]
), T. brucei (J03157
[GenBank]
),
Mastigamoeba invertens (AF083338
[GenBank]
), Bonnemaisonia hamifera
(U90209
[GenBank]
), T. vaginalis (U20501
[GenBank]
), and G. lamblia and Rpc1
sequences of G. lamblia, S. cerevisiae, and H. sapiens were
entered in FASTA format at the MEME web site
(meme.sdsc.edu/meme/website/meme.html)
(40). To restrict the
alignment to the -amanitin motif, the training sequences included the
amanitin motif described for M. musculus, D. melanogaster, C.
elegans, and S. cerevisiae
(41) The multiple sequence
alignment (MSA) of these orthologs was also done using ClustalW
(42), which allows gaps to be
introduced in the sequences. The results were identical to that of the MEME
output within the amanitin motif. A similar procedure was followed to obtain a
block diagram of the bridge helix.
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RESULTS |
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Frequent antisense transcripts for a variety of genes have been reported in
G. lamblia (45,
46); therefore, we used
complementary 60-mer antisense and sense oligonucleotides for the tubulin gene
to distinguish between sense and antisense transcripts in the run-on product.
The antisense probe gave a strong signal, whereas the sense probe gave no
signal (Fig 1A),
indicating that the level of antisense transcription for the tubulin gene is
low or absent. Control experiments using RNase A before hybridization yielded
no signal, as expected (data not shown). We observed no inhibition of
transcription in the presence of 50 µg/ml amanitin with the tubulin,
giardin, or the vsp conserved region probes
(Fig 1A). RNAP I
transcription as measured by hybridization to the rRNA gene was unaffected at
a concentration of 50 µg/ml. In contrast to the amanitin resistance of
Giardia RNAP II, transcription of the S. cerevisiae
EF1- and tubulin genes was almost totally inhibited by 50 µg/ml
amanitin, whereas rRNA transcription was unaffected
(Fig 1B). Actinomycin
D almost completely inhibited G. lamblia RNAP I and RNAP II
transcription at a concentration of 10 µg/ml
(Fig. 2).
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Amanitin Susceptibility of RNAP III TranscriptionBecause eukaryotic RNAP III transcription is partially susceptible to high concentrations of amanitin, we used the leucyl-tRNA gene to evaluate the sensitivity of Giardia RNAP III transcription to amanitin. A putative G. lamblia leucyl-tRNA sequence from the BLAST annotations of the genomic sequences was 78.6% identical to the C. elegans ortholog (Fig. 3). A complementary oligonucleotide identified an 80-nucleotide band on a Northern blot (Fig. 4), which is the expected size of a tRNA gene. Run-on assays at varying concentrations of amanitin (50, 250, and 1000 µg/ml) using the leucyl-tRNA antisense oligonucleotide probe demonstrated that RNAP III transcription was 85% inhibited by 50 µg/ml of amanitin, whereas RNAP I and RNAP II transcription was unaffected (Fig. 5). When we separated the run-on transcripts on a denaturing polyacrylamide gel, we observed a series of bands ranging in size from 70 to 82 nucleotides (Fig. 6), which most likely represent tRNA, as well as 93- and 110-nucleotide bands, which were present in the amanitin untreated sample and not in the treated ones. We do not know the identity of the 93- and 110-nucleotide bands.
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Identification of G. lamblia rpb GenesTo evaluate the potential molecular basis for amanitin resistance as well as other differences of G. lamblia RNAP II transcription from that of other eukaryotes, we identified the genes that encode the subunits of RNAP II from the Giardia genome data base. We identified rpb1, rpb2, rpb3, rpb5, rpb6, rpb7, rpb8, rpb10, rpb11, and rpb12, including two isoforms of rpb5 (rpb5a and rpb5b), but did not find rpb4 or rpb9. However, we did identify rpc9, which demonstrated a 23% identity with the S. cerevisiae Rpb9 along almost the entire length of the polypeptide, including the cysteine residues that coordinate Zn2+ ions in both Rpb9 and Rpc9. Table I shows the chromosomal location and approximate transcript sizes for each of the rpb genes. The rpb genes are distributed among all five chromosomes, and restriction digests (data not shown) show that each is present as a single copy. As for most G. lamblia genes, the transcripts of the rpb genes were approximately the same size as the ORFs (Table I). The comparisons of the G. lamblia and S. cerevisiae Rpb amino acid sequences are shown in Table I.
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Molecular Correlations between the Rpb1 Sequence and Amanitin
Resistance in G. lamblia and Other Organisms Amanitin contacts
residues in two helices in Rpb1, the amanitin motif (residues 719779 in
S. cerevisiae) and the bridge helix (residues 810825), as
shown by -amanitin-RNA polymerase II co-crystals at 2.8-Å
resolution (47). The residues
of the bridge helix of Rpb1 directly contact the DNA base that is paired with
the first base in the RNA strand, whereas the amanitin motif forms a helix
that lines a funnel-shaped cavity through which nucleotides gain access to the
active site. The amanitin-RNAP II co-crystals show potential hydrogen bonds
between amanitin and S. cerevisiae residues 722 (leucine) and 769
(serine) (48), which are also
conserved in other eukaryotes. Therefore, we analyzed this region with the MSA
tool MEME (40)
(Fig. 7). These positions are
shown at positions 5 and 52 on Fig.
7 and correspond to the mouse R749P/Drosophila R741H and
mouse N792D mutations that are associated with amanitin resistance
(41). In G. lamblia,
valine replaces the leucine at position 5, whereas phenylalanine replaces
asparagine at position 52. Other potential hydrogen bonds have been suggested
between amanitin and the glutamine residues at positions 50 and 51. These
glutamine residues are conserved in all the Rpb1 orthologs including that of
G. lamblia (Fig.
7).
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Four additional mutations in the amanitin motif associated with amanitin
resistance have been described
(41). Replacement of cysteine
with tyrosine at position 47 (Fig.
7) is associated with resistance in C. elegans (C777Y)
(41), possibly because of the
presence of the hydroxyl group of tyrosine rather than its bulky nature, since
T. brucei has a phenylalanine at the same position but is
-amanitin-sensitive. This position is occupied by serine in G.
lamblia. The R749P mutation (position 9 in
Fig. 7) confers
-amanitin resistance in M. musculus, and in G.
lamblia this position is occupied by serine. The
-amanitin-resistant G55E mutation has been reported in C.
elegans. This glycine is conserved in all the Rpb1 sequences except
G. lamblia, which has a serine residue at that position. Replacement
of isoleucine with phenylalanine at position 39 in M. musculus has
also been associated with amanitin resistance. G. lamblia has a
phenylalanine at this position. Thus, the amanitin motif of G.
lamblia Rpb1 differs from that of other eukaryotes at all six conserved
positions that have been associated with amanitin resistance in other
organisms.
The Rpc1 amanitin motif sequences of G. lamblia, S. cerevisiae, and H. sapiens contained five of the six conserved amino acids. All the three Rpc1 orthologs had substitutions at position 52 of the amanitin motif (Fig. 7).
The movement of the bridge helix is important for the translocation of RNAP II on DNA, and binding of amanitin to the bridge helix imposes a constraint on its movement, hampering the translocation of the polymerase on DNA. X-ray co-crystals showed a very strong hydrogen bond between S. cerevisiae glutamate residue 822 of the bridge helix and amanitin (47). This glutamate is conserved in all Rpb1 orthologs with the exception of T. vaginalis, which has a threonine residue at that position (Fig. 8). Therefore, it is possible that this substitution contributes to the amanitin resistance observed in T. vaginalis RNAP II. The glutamate is conserved in RNAP III (Rpc1) of S. cerevisiae and H. sapiens, whereas the G. lamblia sequence encodes a conservative substitution (aspartate).
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Other Comparisons of G. lamblia RNAP II with Other EukaryotesRpb1 and Rpb2 form the two lobes flanking the cleft through which the DNA gains entry to the active site. The active site of the enzyme is marked by the presence of two Mg2+ ions, designated A and B (47). The metal ion A is coordinated by three alternating aspartate residue side chains present in an absolutely conserved motif, NADFDGD, at positions 479485 in S. cerevisiae Rpb1 (47, 49). This motif is found at positions 496502 of the G. lamblia Rpb1 and is in perfect alignment with the motif in all the other eukaryotes in the MSA generated using ClustalW with a BLOSUM62 substitution model (data not shown). The metal ion B at the active site is held in correct position for the formation of the phosphodiester bond by glutamate and aspartate residue side chains at positions 835 and 836, respectively, in the yeast Rpb2 (47, 50). These residues are conserved in all eukaryotic Rpb2 orthologs and are present at positions 877 and 878 in the G. lamblia ortholog in our MSAs (data not shown).
The C-terminal domain is required for enhancer-driven transcription and consists of heptapeptide repeats (consensus YS-TPSTS) in most Rpb1 orthologs. The C-terminal domain also recruits factors involved in co-transcriptional processes, such as 5' capping, splicing, and transcription termination. The hydroxyl side chains of the C-terminal domain are hyperphosphorylated during elongation and hypophosphorylated during initiation of transcription. Most protists including G. lamblia lack regular repeats but have a frequent occurrence of hydroxyl-containing amino acids in this domain (Table II).
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Nucleotide triphosphates gain entry through a pore (known as pore1) in RNAP II beneath the active site. The pore module has a conserved motif PXIXKPXXLWXGKQ (where X is any amino acid), which is essential for the interaction of Rpb1 with Rpb8 (51). The sequence of the G. lamblia Rpb1 pore module is PXIXYPXXRWXGKQ, beginning at position 689. The rpb8 motif that interacts with rpb1, GGLLM, at position 119 in the yeast is replaced by GGLIA at position 120 in the G. lamblia rpb8 gene.
In S. cerevisiae, Rpb5 and Rpb9 (along with some regions of Rpb1) flank the Rpb1-Rpb2 cleft and position the downstream DNA. We identified two isoforms of Rpb5 in G. lamblia using the TBLASTN function with S. pombe Rpb5 as the query sequence. They are 229 and 201 amino acids long and positions 136229 of Rpb5a and 108201 of Rpb5b are 43% identical, whereas there is little similarity throughout the remainder of the genes. Both G. lamblia Rpb5 genes align with S. cerevisiae ortholog over nearly the entire sequence and are 30% identical and 46% similar (Rpb5a) or 26% identical and 41% similar (Rpb5b) to the S. cerevisiae ortholog. Thus, in terms of overall alignment, the two G. lamblia Rpb5s are nearly as similar to S. cerevisiae Rpb5 as to each other. Proline residues at positions 86 and 118 of S. cerevisiae Rpb5 contact the DNA backbone through nonspecific van der Waals interactions, which might favor a particular rotational setting for DNA while allowing its helical screw rotation (50). Neither proline residue was present in either of the G. lamblia Rpb5 sequences.
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DISCUSSION |
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Our inability to detect rpb4 and rpb9 may indicate their absence from the genome, a level of divergence making them impossible to detect by BLAST or Hidden Markov Models (the latter attempted for rbp4 as available at pfam.wustl.edu), or could indicate that they have not yet been sequenced. Because the genome project is now at 7.7-fold coverage, we expect that 99% of the genome has been sequenced, making it relatively unlikely that these genes have not been sequenced. It is notable that these are the only two subunits that are nonessential in S. cerevisiae, lending credibility to the possibility that these subunits are truly absent in Giardia.
Mutations in Rpb9 affect the start site of transcription (5254) but are not lethal for yeast. Because G. lamblia has very short 5'-untranslated regions and lacks the typical eukaryotic RNAP II promoters, it is possible that Rpb9 is not required. Alternatively, it is possible that another protein such as Rpc9 performs that function in G. lamblia. In contrast to the apparent absence of two rpb genes, we found two rpb5 genes. The reason for this finding is not clear.
There is considerable controversy regarding whether the cellular, molecular, and biochemical differences between G. lamblia and other eukaryotes are ancestral or whether they represent a more recent divergence from other eukaryotes. A better understanding of transcription in Giardia along with its unique features may help to resolve this controversy. In addition, it is possible that the difference in the structure of the G. lamblia RNAP II that leads to amanitin resistance can be exploited for the development of novel chemotherapeutic agents.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants
AI43273 (to M. L. S.) and AI51089 (to A. G. M.). Additional support was
provided by the G. Unger Vetlesen Foundation and LI-COR Biotechnology. The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
|| To whom correspondence should be addressed: Dept. of Microbiology/Immunology, University of Arizona, 1501 N. Campbell, Tucson, AZ 85724-5049. Tel.: 520-626-6430; Fax: 626-2100; E-mail: adamr{at}u.arizona.edu.
1 V. Seshadri and R. D. Adam, unpublished results.
2 The abbreviations used are: RNAP, RNA polymerase; MSA, multiple sequence
alignment; MEME, multiple expectation maximizations and motif elicitation;
contig, group of overlapping clones; ORF, open reading frame.
3 V. Seshadri, A. G. McArthur, M. L. Sogin, and R. D. Adam, unpublished data
from MEME (multiple expectation maximizations and motif elicitation) analysis
of 14 published vsp genes.
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ACKNOWLEDGMENTS |
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REFERENCES |
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