Two Small Subunits in Arabidopsis RNA Polymerase II Are Related to Yeast RPB4 and RPB7 and Interact with One Another*

An Arabidopsis cDNA (AtRPB15.9) that encoded a protein related to the RPB4 subunit in yeast RNA polymerase II was cloned. The predicted molecular mass of 15.9 kDa for the AtRPB15.9 protein was significantly smaller than 25 kDa for yeast RBP4. In SDS-PAGE, AtRPB15.9 migrated as the seventh or eighth largest subunit (i.e. apparent molecular mass of 14–15 kDa) inArabidopsis RNA polymerase II, whereas RPB4 migrates as the fourth largest subunit (i.e. apparent molecular mass of 32 kDa) in yeast RNA polymerase II. Unlike yeast RPB4 and RPB7, which dissociate from RNA polymerase II under mildly denaturing conditions, plant subunits related to RPB4 and RPB7 are more stably associated with the enzyme. Recombinant AtRPB15.9 formed stable complexes with AtRPB19.5 (i.e. a subunit related to yeast RPB7) in vitro as did recombinant yeast RPB4 and RPB7 subunits. Stable heterodimers were also formed between AtRPB15.9 and yeast RPB7 and between yeast RPB4 and AtRPB19.5.

[1]  A. Sentenac,et al.  Two forms of RNA polymerase B in yeast. Proteolytic conversion in vitro of enzyme BI into BII. , 1976, European journal of biochemistry.

[2]  A. Sentenac,et al.  A mutation of the B220 subunit gene affects the structural and functional properties of yeast RNA polymerase B in vitro. , 1980, The Journal of biological chemistry.

[3]  R. Burgess,et al.  Studies of the subunit structure of wheat germ ribonucleic acid polymerase II. , 1977, Biochemistry.

[4]  R. Young,et al.  RPB7, one of two dissociable subunits of yeast RNA polymerase II, is essential for cell viability , 1993, Yeast.

[5]  F. Studier,et al.  Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. , 1986, Journal of molecular biology.

[6]  R. Ebright,et al.  Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  R. Burgess,et al.  Large-scale purification of wheat germ DNA-dependent RNA polymerase II , 1975 .

[8]  E. Stellwagen.  [25] Gel filtration , 1990 .

[9]  S. Jacob.  Enzymes of nucleic acid synthesis and modification , 1983 .

[10]  F. Regad,et al.  cDNA cloning and expression of an Arabidopsis GTP‐binding protein of the ARF family , 1993, FEBS letters.

[11]  R. Young,et al.  A portion of RNA polymerase II molecules has a component essential for stress responses and stress survival , 1993, Molecular and cellular biology.

[12]  T. Boulikas,et al.  Silver staining of proteins in polyacrylamide gels. , 1981, Analytical biochemistry.

[13]  T. Guilfoyle,et al.  Reconstitution of Yeast and Arabidopsis RNA Polymerase α-like Subunit Heterodimers* , 1997, The Journal of Biological Chemistry.

[14]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[15]  D. Botstein,et al.  Human RNA polymerase II subunit hsRPB7 functions in yeast and influences stress survival and cell morphology. , 1995, Molecular biology of the cell.

[16]  D. Reinberg,et al.  The nonphosphorylated form of RNA polymerase II preferentially associates with the preinitiation complex. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[17]  T. Guilfoyle,et al.  Eukaryotic RNA polymerase: comparative subunit structures, immunological properties, and alpha-amanitin sensitivities of the class II enzymes from higher plants. , 1978, Biochemistry.

[18]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[19]  R. Young,et al.  Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro. , 1991, The Journal of biological chemistry.

[20]  P. Moore,et al.  Six human RNA polymerase subunits functionally substitute for their yeast counterparts , 1995, Molecular and cellular biology.

[21]  T. Guilfoyle,et al.  Plant DNA-dependent RNA polymerases: subunit structures and enzymatic properties of the class II enzymes from quiescent and proliferating tissues. , 1978, Biochemistry.

[22]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[23]  N. Woychik,et al.  Functional substitution of an essential yeast RNA polymerase subunit by a highly conserved mammalian counterpart , 1994, Molecular and cellular biology.

[24]  G. V. Shpakovski,et al.  Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[25]  A. Ishihama,et al.  Subunit composition of RNA polymerase II from the fission yeast Schizosaccharomyces pombe. , 1996, Gene.

[26]  M. Choder.  A growth rate-limiting process in the last growth phase of the yeast life cycle involves RPB4, a subunit of RNA polymerase II , 1993, Journal of bacteriology.

[27]  T. Guilfoyle,et al.  Association between 36- and 13.6-kDa -Like Subunits of Arabidopsis thaliana RNA Polymerase II (*) , 1996, The Journal of Biological Chemistry.

[28]  R. Young,et al.  RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth , 1989, Molecular and cellular biology.

[29]  J. Acker,et al.  Interactions between the Human RNA Polymerase II Subunits* , 1997, The Journal of Biological Chemistry.