Elsevier

Gene

Volume 187, Issue 2, 18 March 1997, Pages 165-170
Gene

Isolation and characterization of cDNA encoding mouse RNA polymerase II subunit RPB14

https://doi.org/10.1016/S0378-1119(96)00739-1Get rights and content

Abstract

By means of the yeast two-hybrid system using the 40-kDa subunit of mouse RNA polymerase I, mRPA40, as the bait, we isolated a mouse cDNA which encoded a protein with significant homology in amino acid sequence to the 12.5-kDa subunit of Saccharomyces cerevisiae RNA polymerase II, B12.5 (RPB11). Specific antibody raised against the recombinant protein that was derived from the cDNA reacted with a 14-kDa polypeptide in highly purified mammalian RNA polymerase II and did not react with any subunit of RNA polymerase I or III. Moreover, the antibody co-immunoprecipitated the largest subunit of mouse RNA polymerase II. These results provide biochemical evidence that the cDNA isolated, named mRPB14, encodes a specific subunit of RNA polymerase II, and indicate that the subunit organization of the enzyme is conserved between yeast and mouse. A possible role of the α-motif [Dequard-Chablat, M., Riva, M., Carles, C. and Sentenac, A., J. Biol. Chem. 266 (1991) 15300–15307] in the protein-protein interaction between mRPA40 and mRPB14 is also discussed.

Introduction

A single type of RNA polymerase mediates essentially all RNA synthesis in prokaryotes. The Escherichia coli RNA polymerase holoenzyme is composed of two α, β, β′, and σ subunits. The α subunit plays the key role in subunit assembly, since homo-dimerization of the α subunit is the first step in the pathway to form the bacterial core-enzyme α2ββ′ (Yura and Ishihama, 1979). In contrast, three distinct nuclear RNA polymerases transcribe different sets of genes in eukaryotic cells. Pol I synthesizes rRNA precursors, Pol II transcribes protein-coding genes, and Pol III produces 5S rRNA, small nuclear RNAs and tRNAs. The subunit composition of the eukaryotic polymerases has been extensively characterized in yeast Saccharomyces cerevisiae; each class of the polymerases is composed of two large polypeptides (>100 kDa) and 10–14 smaller subunits (<85 kDa) (Sentenac et al., 1992; Young, 1991). The two largest subunits of each class of the enzyme have homology to the bacterial β′ and β subunit, respectively. Among the smaller subunits, two distinct ones have been shown to possess an aa stretch conserved in the bacterial α subunit, i.e., B44.5 (RPB3) and B12.5 (RPB11) for yeast Pol II, and AC40 and AC19 for Pol I and Pol III of yeast (Dequard-Chablat et al., 1991; Kolodziej and Young, 1989; Mann et al., 1987; Woychik et al., 1993). These α-related subunits seem to play an important role in subunit assembly (Kolodziej and Young, 1989; Lalo et al., 1993; Ebright and Busby, 1995). Therefore, in eukaryotic cells, heterodimer formed between the two α-related subunits may play an analogous role to the bacterial α-subunit homodimer.

Recent isolation of cDNAs encoding human Pol II subunits showed that its subunit composition is fundamentally the same as yeast Pol II (Shpakovski et al., 1995). In contrast, however, the structures of mammalian Pol I and Pol III largely remain to be determined. To elucidate the structure and function of mammalian Pol I, we have previously purified mouse Pol I to homogeneity and subsequently isolated cDNA encoding the 40-kDa subunit based on the peptide sequences obtained from the purified enzyme (Song et al., 1994). The aa sequence from the cDNA clone showed high similarity with yeast AC40 subunit, leading us to the conclusion that the 40-kDa subunit, named mRPA40, is a mouse homolog of yeast AC40. It remains to be determined whether mRPA40 is a common subunit shared by mouse Pol I and Pol III. To extend our knowledge in the structure and function of mammalian Pol I subunits, we screened mouse cDNA library to isolate mRPA40-interacting proteins by yeast two-hybrid system. Among the candidate isolates, two clones showed sequence similarity with yeast AC19 and B12.5 subunit, respectively. In this paper, we report the characterization of one of the clones, mRPB14, the mouse homolog of yeast B12.5 subunit.

Section snippets

Isolation of cDNA encoding mRPA40-interacting proteins

The yeast two-hybrid system (Chien et al., 1991) was used for screening proteins which might interact with mRPA40 (Song et al., 1994). Yeast strain Y153 (Durfee et al., 1993) was transformed first with a plasmid which produces a fusion protein between GAL4(DB) and mRPA40 [GAL4(DB)/mRPA40], and subsequently with an expression library of mouse embryonic cDNAs fused to the GAL4(TA) region (Chevray and Nathans, 1992). Formation of a complex between the GAL4(DB)/mRPA40-fusion protein and a protein

Conclusions

  • 1.

    A mouse cDNA encoding a 117-aa polypeptide was isolated by two-hybrid system using a mouse Pol I subunit mRPA40 as the bait. The protein encoded by this cDNA was proved biochemically to be a specific subunit of Pol II, and was named mRPB14.

  • 2.

    A critical aa residue for protein-protein interaction in α-motif has been shown to be conserved between mRPA40 and yeast AC40 subunit. The mRPA40 can interact with mRPB14 through its α-motif in the yeast two-hybrid system.

  • 3.

    The aa sequence comparisons suggest

Acknowledgements

We wish to thank Drs. P.M. Chevray and D. Nathans for the plasmid pPC97 and the mouse cDNA library, T. Chibazakura and S. Kitajima for the purified Pol II, and Z. Wang and R.G. Roeder for the purified Pol III. We also thank A. Okuda, K. Hisatake, M. Nishimoto, A. Orimo, R. Yamada and A. Fukushima for helpful discussions, and N. Hihara for excellent technical assistance. This work was supported by Grants-in-Aid for Ministry of Education, Science and Culture of Japan.

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