Abstract
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.
Highlights
Higher plant plastid ribosomes are closely related to those found in eubacteria, reflecting the endosymbiotic origin of chloroplasts
Proteins from the first 1 M NaCl elution step of the last column were transferred to Immobilon-P membranes and tested either with preimmune serum, with antibodies raised against total 50 and 30 S ribosomal proteins or with antibodies raised against T3 RNA polymerase. From this result we can conclude that CDF2 co-purifies with the T7-like plastid RNA polymerase and two r-protein-like polypeptides on three different columns
To analyze whether the intron in the region of the highly acidic extension is conserved between species, we screened a genomic library of Arabidopsis thaliana, ecotype Columbia, that was cloned in GEM11 using the cDNA as a probe
Summary
Higher plant plastid ribosomes are closely related to those found in eubacteria, reflecting the endosymbiotic origin of chloroplasts. From this result we can conclude that CDF2 co-purifies with the T7-like plastid RNA polymerase and two r-protein-like polypeptides on three different columns. Cloning, Sequencing, and Characterization of the cDNA and Gene for One of the Two Supposed r-proteins—The two polypeptides that cross-reacted with the r-protein antibodies were cut out and their N-terminal amino acid sequences were determined.
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