Poly(A) Polymerase and Poly(G) Polymerase in Wheat Chloroplasts

Abstract

Extracts of wheat chloroplasts contain a poly(A) polymerase which can polymerize AMP residues from ATP onto an RNA primer. Whole extracts of wheat leaves also contain another poly(A) polymerase which is present in much larger amount and is probably derived from the nuclei. Both polymerases can utilize as primer poly(A), poly(C), transfer RNA, and ribosomal RNA, but only the chloroplast polymerase can utilize poly(U) and poly(G). Both enzymes have a specific requirement for ATP. Extracts of wheat chloroplasts contain, in addition to the poly(A) polymerase, a poly(G) polymerase which can polymerize GMP residues from GTP onto primers such as poly(G), poly(A), or ribosomal RNA. The poly(G) polymerase cannot utilize ATP but can slowly polymerize CMP from CTP. When the two chloroplast polymerases are present together in an in vitro incubation with ATP plus GTP and poly(A), the polymerization product is a mixed poly(A,G) tract.

The Mechanism of Preferential Degradation of Polyadenylated RNA in the Chloroplast

“…However, depletion of 100RNP/PNPase from the chloroplast extract by heparin or single-stranded DNA columns did not interfere with in vitro polyadenylation activity (27). Moreover, a poly(A) polymerase has been isolated from chloroplast extract (37). 3 A similar situation of in vitro polymerase activity of the PNPase and the existence of poly(A) polymerases was reported for bacteria (14).…”

“…However, we had previously shown that depletion of 100RNP/PNPase from the chloroplast protein extract did not interfere with in vitro polyadenylation activity (27). In addition, a poly(A) polymerase had been purified from spinach chloroplast extract (37). 3 If the chloroplast 100RNP/PNPase activity is modulated similar to the E. coli PNPase, then the concentrations of inorganic phosphate and nucleotide diphosphate in the chloroplast would promote the degradation rather than the polymerization activity.…”

Plant mRNA 3?-end formation

“…Although there is not enough direct nucleotide sequence homology to use either the mammalian or yeast PAP clones as molecular probes to isolate a plant homologue (unpublished data), PAP is the one component of the processing machinery 55 which can be purified without the need for a functional 3t-processing extract. In fact, PAP activities in plant tissues were first reported over twenty years ago [ 13,79,103,111], and have been described in a diverse range of plants such as cotton [46], maize [78], wheat [13,66,68,111], pea [7], mung bean [80], cow pea [120,121 ], and tobacco [22]. Plant PAPs have been purified to apparent electrophoretic homogeneity from mung bean hypocotyls [106], germinating wheat embryos [65], and cow pea seedlings [120].…”

Polyadenylation and Degradation of mRNA in the Chloroplast1

“…Furthermore, the guanosine residues characteristic of the poly(A) tails of chloroplast psbA mRNA may be involved in modulating the activity of the respective enzyme. On the other hand, they may simply reflect the specificity of the poly(A) polymerase(s) (Burkard and Keller, 1974;Lisitsky et al, 1996). In vitro experiments in which synthetic transcribed RNA with poly(A) tails of different lengths and different proportions of guanosine residues were incubated with chloroplast protein extract revealed remarkable differences in degradation rates.…”

“…In vitro analysis of chloroplast polyadenylation activity revealed specificity to ATP and GTP, reflecting the composition of the poly(A)-rich tails observed by RT-PCR described above. In this respect, it is interesting to note that poly(A)-and poly(G)-polymerase activities were purified from wheat chloroplasts 25 years ago (Burkard and Keller, 1974). Furthermore, in vitro polyadenylation activity is dependent on the substrate structure.…”