Genes for Nuclear RNA Polymerases
Overview
Useful attributes of these genes are:
- Large size, providing many characters, about 4000bp for the largest and 3000bp for the second largest subunit.
- Convenient amplification from conserved PCR primers (see below)
- Sequences that are easily alignable; particularly for RPB2 and RPC2. Typically, the amount that needs to be excluded because of ambiguities in alignment is much less than for ssu or lsu rRNA.
- Rates of evolutionary change that are convenient either for species level or deep phylogenetic work, depending on the segment used. The subunits of Pol I have evolved 2x to 3x faster than those of Pols II and III.
- Intron sequence ranging in length from less than 100bp to greater than 1000bp. Sequences of the longer introns can provide excellent phylogenetic resolution between close relatives. For example, see Goetsch et al. Sys. Bot. 30(3): 616-626 (2005).
Gene Names
The eukaryotic nuclear RNA polymerases were discovered almost simultaneously in North America and Europe. Initially, the American workers named the enzymes Pol I, II, and III while the French referred to them as A, B, and C. By now, the I, II, III nomenclature is well established for the polymerases, whereas the genes encoding their subunits are named according to the A, B, C system. RPB1 (largest subunit of pol II), RPC1 (largest subunit of pol III), RPB2 (second-largest subunit of pol II), etc.
Gene Structure for RPB1 and RPB2
In addition to sequences coding for RNA polymerase subunits, these genes contain introns in phylum-specific patterns.
Rhodophytes: These genes have an occasional intron, which invariably occurs near the N-terminus.
Fungi: Introns are rare in Ascomycetes; they occur more frequently in Basidiomycetes. As a rule, fungal introns are very short (<<100bp).
Plants: In the Chlorophyte lineage, RPB1 and RPB2 intron positions were established very early. The 13 intron positions of RPB1 and the 24 intron positions of RPB2 are conserved in Zygnematales, Coleochaetales, Charales, and land plants. Only very rarely has an additional intron position been observed. At two junctures in core eudicot evolution, multiple losses of adjacent introns occurred.
Intron-Exon Structure of RPB2 in Green Algae and Land Plants
- Exons 23 and 24 are large and highly conserved in amino acid sequence
- Exons 18-22 are small and also conserved
- Exons 11-17 show large variation in amino acid sequence
- Exons 1-10 are the next most variable region.
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Gene Duplication and Paralogous Gene Copies
While many plants contain a single RPB1 and a single RPB2 copy in the genome, this is not always the case. Defective pseudogenes occur rarely, but both genes are multicopy in certain taxa. Ranunculaceae and Papaveraceae contain two copies of RPB2, which have diverged only slightly. On the other hand, plants in the groups Asteridae I and Ericales both have two genomic copies of RPB2, which are designated RPB2-i and RPB2-d. The two paralogs differ by 10% in DNA sequence for their exons and for the translated amino acid sequence. The d copy is expressed in vegetative tissue and is the only form of RPB2 found in many eudicots (Caryophyllids, Rosids, and Saxifragaceae, for example.) The sequence differences between d and i copies make it possible to design paralog-specific primers to selectively amplifly just RPB2-i or RPB2-d genes (Goetsch et al., 2005).
General RPB2 primers
| RPB2-3bF | (GGYFIING) | 5'-GGW GGW TAY TTY ATY ATY AATGG-3' |
| RPB2-6F | (WGM/LVCPA) | 5'-TGG GGK WTG GTY TGY CCT GC-3' |
| RBP2-6R | (WGM/LVCPA) | 5'-GC AGG RCA RAC CAW MCC CCA-3' |
| RBP2-7F | (MGKQAMG) | 5'-ATG GGK AAG CAR GCW ATG GG-3' |
| RBP2-7R | (MGKQAMG) | 5'-CC CAT WGC YTG CTT MCC CAT-3' |
| RBP2-11aR | (MVDDKIH) | 5'-GTG WAT YTT RTC RTC MAC C-3' |
| RBP2-11bR | (GEMERD) | 5'-CA ATC WCG YTC CAT YTC WCC-3' |
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RPB2 genes in Rhododendron: the Diagram shows the locations of introns in RPB2
(the primers below are named for their exon locations)
Rhododendron-specific PCR Primers for RPB2-i
| RPB2- 2F (RNLTYSSPL) | 5'-G AAC CTG ACC TAC TCA TCT CCA TT-3' |
| RPB2-3R (PNKYAYVSE) | 5'-TC GGA TAC ATA AGC ATA TTT GTT GG-3' |
| RPB2-3F (PNKYAYVSE) | 5'-CC AAC AAA TAT GCT TAT GTA TCC GAG-3' |
| RPB2-4R (FRALGFVAD) | 5'-TC TGC AAC AAA TCC CAG TGC ACG G-3' |
| RPB2-4F (FRALGFVAD) | 5'-C CGT GCA CTG GGA TTT GTT GCA GA-3' |
| RPB2-6R (GTGEYCET) | 5'-AGT TTC ACA ATA TTC CCC CGT G-3' |
| RPB2-6F (GTGEYCET) | 5'-C ACG GGG GAA TAT TGT GAA ACT-3' |
| RPB2-12R (PILEFLEE) | 5'-CTC CTC CAG AAA CTC CAA TAT G-3' |
| RPB2-13F (IHRNPEL) | 5'-TT CAT CGT AAC CCT GAG CTC-3' |
| RPB2-16R (TSARLCP) | 5'-TGG GCA GAG ACG TGC AGA TG-3' |
| RPB2-23F (TIEGICPDI) | 5'-A ATT GAG GGC ATC TGT CCA GAC ATC-3' |
| RPB2-24R (TGRPLTYTI) | 5'-T CGT ATA AGT CAG CGG ACG CCC TG-3' |
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References: Phylogeny based upon RPB1 and RPB2 Sequences
Metazoans (RPB1 and RPB2)
Sidow, A. and W.K. Thomas (1994) Curr. Biol. 4: 596-603.
Protists (RPB1)
Stiller, J.W. and B.D. Hall (1997). Proc. Nat. Acad. Sci. USA 94: 4250-4525.
Plants (RPB2)
Denton, A.L., B.L. McConaughy, and B.D. Hall (1998) Mol. Biol. Evol. 15: 1082-1085.
Oxelman, B., N. Yoshikawa, B.L. McConaughy, , J. Luo, A.L. Denton, , and B.D. Hall, (2004) Mol. Phylo. Evol. 32: 462-479.
Goetsch, L., A. J. Eckert, and B. D. Hall (2005). Sys. Bot. 30(3) 616-626.
Plants (RPB1)
Hajibabaei, M., J. Xia and G. Drouin (2006). Mol. Phylo. Evol. 40(1):208-217.
Ascomycetes (RPB2)
Liu, Y.J., Whelen, S. and B.D. Hall (1999). Mol. Biol. Evol. 16: 1799-1808.
Liu Y.J. and B. D. Hall (2004) Proc. Nat. Acad. Sci. USA. 101:4507-4512.
Basidiomycetes (RPB1 and RPB2)
Matheny, P.B., Liu, Y.J., Ammirati, J.F. and B.D. Hall (2002). Am. J. Bot. 89:688-698.
Matheny, P.B. Mol. Phylo. Evol. (2005). 35(1) :1-20.
Microsporidians in relation to fungi (RPB1)
Hirt, R.P. et al. (1999) Proc. Nat. Acad. Sci. USA 96: 580-585.