By means of mobility-shift assays and Exonuclease III mapping we have determined a 14 bp sequence (named CDF2 binding site) located in front of the 16S rRNA initiation start site which is protected by a spinach chloroplast extract. This region does not include neither one of the two '-35' nor of the two '-10' E. coli-like promoter elements which are recognised by E. coli RNA polymerase in vitro. The CDF2 binding site is specifically recognized by two small polypeptides which migrate corresponding to 35 and 33 kDa respectively as shown by UV cross-linking experiments. In vivo transcription initiation of the 16S rRNA gene occurs 13 nucleotides downstream of the 14 bp sequence and is different from the transcription start site which is used by E.coli polymerase in vitro.
Spinach chloroplasts contain two types of RNA polymerases. One is multimeric and Escherichia coB-like. The other one is not E. coli-like and might represent a monomeric enzyme of 110 kD. The quantitative relation of the two polymerases changes during plant development. This raises the question, how are plastid genes transcribed that contain E. coli-like and non-E, coil-like promoter elements during developmental phases when both enzymes are present? Transcription of the spinach plastid rrn operon promoter is initiated at three sites: P1, PC, and P2. P1 and P2 are preceded by E. coli-like promoter elements that are recognized by E. coil RNA polymerase in vitro. However, in vivo, transcription starts exclusively at PC. We analyzed different promoter constructions using in vitro transcription and gel mobility-shift studies to understand why Pl and P2 are not used in vivo. Our results suggest that the sequence-specific DNA-binding factor CDF2 functions as a repressor for transcription initiation of the E. coli-like enzyme at PI and P2. We propose a mechanism of constitutive repression to keep the rrn operon in all developmental phases under the transcriptional control of the non-E, coli-like RNA polymerase.
We have cloned and sequenced the cDNA and the gene coding for plastid ribosomal protein L4 (RPL4) from two higher plant species, spinach and Arabidopsis thaliana. Ribosomal protein L4 is one of the ribosomal proteins for which extraribosomal functions in transcriptional regulation has been demonstrated in prokaryotes. Sequence comparison of the two plant cDNAs and genes shows that the RPL4 gene has acquired a remarkable 3 extension during evolutionary transfer to the nuclear genome. This extension harbors an intron and codes for a glutamic and aspartic acid-rich amino acid sequence that resembles highly acidic C-terminal tails of some transcription factors. Co-purification of ribosomal protein L4 with plastid RNA polymerase and transcription factor CDF2 using different purification protocols as well as the surprising amino acid sequence of the L4 protein make it a likely candidate to play a role in plastid transcriptional regulation.Higher plant plastid ribosomes are closely related to those found in eubacteria, reflecting the endosymbiotic origin of chloroplasts. They contain about 54 -75 ribosomal proteins, depending on the plant species (1-3). The complete sequencing of several higher plant plastid genomes has shown that about one third of the plastid ribosomal (r) 1 proteins are encoded by the plastid genome itself (4 -7). The remaining two thirds are probably encoded by the nuclear genome, and it is thought that the genes have been transferred from the plastid genome to the nucleus during evolution. From these nuclear-encoded plastid r-proteins, only few have been characterized by their cDNAs and/or by their genes (see Harris et al. (1) and references therein).During the last 10 years ribosomal proteins became of special interest because it was shown in a number of cases that they have extraribosomal functions apart from the ribosome and protein biosynthesis (for review, see Wool (8)). These extraribosomal functions concern basic cellular processes like replication, transcription, RNA processing, translation, and DNA repair. Specific functions in transcription have been reported for r-proteins S10 (9 -12), L4 (13-15) and S14 (16). In all three cases, the regulation by r-proteins concerns the expression of ribosomal components (rRNA, operon S10, and mRNA for rprotein S14).The plastid genome is transcribed by two different RNA polymerases. One is nuclear-encoded, T7-like, and especially active during early phases of plastid development. This polymerase is more or less specified for transcription of plastid housekeeping genes, encluding subunits of the second RNA polymerase, which is plastid-encoded, prokaryotic-like, and transcribes preferentially photosynthesis-related genes during later phases of plastid development (17-21). The transcriptional activity of the prokaryotic-type RNA polymerase is regulated by nuclear-encoded, prokaryotic-type, and sigma-like factors (22, 23).Our group has been working for several years on the expression of rrn transcription in spinach plastids. Transcription of the rrn opero...
A photosynthetic mutant of Arabidopsis thaliana, hcf5, was isolated by screening M, seedlings for high chlorophyll fluorescence. Thylakoid morphology was strikingly abnormal, with large grana stacks and almost no stroma lamellae. Fluorescence induction kinetics, activity assays, and immunoblotting showed that photosystem II was absent. Polypeptides of the photosystem I complex, the Cyt b d f complex, coupling factor, and the large subunit of ribulose-1,s-bisphosphate carboxylase/oxygenase were also severely depleted. However, the nuclear-encoded chlorophyll a/b lightharvesting complex polypeptides were unaffected. The rbcL transcript was present at very low levels, the pattern of transcripts from the polycistronic psbB-psbH-petB-petD operon was abnormal, and the mature psbH message was almost completely lacking. This suggests that the hcf5 locus may encode a product required for the correct expression of several chloroplast genes.
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