Nascent transcript-binding protein of the pea chloroplast transcriptionally active chromosome.

This study describes the nascent RNA-binding protein of the pea chloroplast transcriptional complex. The protein has been identified by photoaffinity labelling of the transcriptionally active chromosome (TAC) which utilizes the endogenous plastid DNA as template. UV irradiation of lysed chloroplast or the isolated TAC under conditions optimized for transcription photocross-links nascent radiolabelled transcripts (up to 250 nucleotides in length) to a 48 kDa protein. The photoaffinity labelling of the transcript-binding protein is dependent on UV irradiation, is maximal after about 30 min of irradiation, and is completely dependent on transcriptional activity; no cross-linkage has been observed with pre-synthesized RNA. Cross-linkage is influenced by salts and inhibitors in accordance with their effects on transcription. The photoconjugate is composed of protein and RNA moieties, and can be hydrolysed by several proteases. However, the cross-linked transcript is protected from nucleases until the protein is removed. Manganese enhances photoaffinity labelling of the transcript-binding protein, and this is paralleled by an increase in total transcriptional activity of TAC. This protein was isolated by 2-dimensional polyacrylamide gel electrophoresis and the sequence of 15 amino acid residues at the amino terminus was determined. The nascent transcript-binding protein appears to be involved in the transcription of all three classes of chloroplast genes. We also found a polypeptide of identical molecular weight to get cross-linked to nascent transcripts in chloroplasts isolated from other legumes such as Cicer arietenum, Vigna radiata and Phaseolus vulgaris, and monocots like Zea mays, Oryza sativa and Pennisetum americanum.

Two distinct transcriptional activities of pea (Pisum sativum) chloroplasts share immunochemically related functional polypeptides.

An RNA polymerase activity has been purified from pea (Pisum sativum) chloroplast extracts with a distinct transcriptional specificity for a chloroplast messenger gene. This activity (ms-RNA pol) differs from the pea RNA polymerase preparation reported by Sun, Shapiro, Wu & Tewari [(1986) Plant Mol. Biol. 6, 429-439], which specifically transcribes only the rRNA gene (rb-RNA pol). The specificity of transcription has been assessed by the synthesis in vitro of discrete transcripts of predicted sizes using cloned promoter regions of the chloroplast psbA and 16 S rRNA genes. The ms-RNA pol preparation, with polypeptides ranging in apparent molecular mass from 22 to 180 kDa, correctly initiates transcription from recombinant plasmids containing the psbA promoter and does not support 16 S rRNA promoter-directed transcription. The two activities differ also in their response to Mn2+ ions. To investigate whether the two transcriptional activities share common functional polypeptides, monoclonal antibodies were developed against the rb-RNA pol preparation. Three clones were selected on the basis of their ability to inhibit transcription in vitro of the 16 S rRNA gene by rb-RNA pol. The antibodies from these clones independently recognized three polypeptides with molecular masses of 27, 90 and 95 kDa on immunoblots. Antibodies cross-reacting with the 90 kDa polypeptide completely eliminated the specific retardation of an end-labelled 16 S rRNA promoter fragment in a mobility-shift assay, whereas the antibodies against the 95 kDa polypeptide resulted in the formation of a ternary complex (enzyme-DNA-antibody). The antibodies cross-reacting with the 27 kDa polypeptide, however, did not alter the mobility of the retarded DNA-enzyme complex on the gel. These antibodies also inhibited transcription in vitro of the psbA gene by ms-RNA pol and recognized polypeptides of identical molecular masses in the ms-RNA pol. These results show that the three polypeptides are functional components of the chloroplast transcriptional complex and appear to be involved in the transcription of both rRNA and mRNA genes. Transcriptional specificity is probably conferred by ancillary transcription factor(s) which remain to be identified.

Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNA in vitro.

We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastid DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and alpha-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thio UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensitivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-protein complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubation of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex.

Characterization of the inhibitory effects of hyperprolactinaemia on the mechanism controlling LH secretion in chronically ovariectomized rats.

The time-course of the inhibitory effect of hyperprolactinaemia on LH secretion was delineated. Hyperprolactinaemia was induced in ovariectomized rats with injections of domperidone or ovine prolactin and circulating LH levels were measured from 1 h to 9 days after the treatment. Inhibition of LH secretion occurred within 2-4 h after treatment, and was maintained (provided that serum prolactin remained elevated) for a period of 6 days only. Thereafter LH levels increased to become insignificantly different from control levels on Day 9. A reduction in pituitary responsiveness was not associated with the acute or sub-chronic inhibition of LH secretion, although a significant fall in responsiveness was observed simultaneously with the return of serum LH levels to control values. No changes in hypothalamic LH-RH content was found. It is concluded that an impairment of pituitary function is not responsible for the inhibitory action of prolactin on LH secretion.