We have determined the sequence of the spinach (Spinacia oleracea) chloroplast genes for the photosystem II proteins, D2 and the 44 kd reaction-centre, chlorophyll a-binding protein, and for tRNASer (UGA). The 3' end of the D2 gene overlaps the first 50 bp of the 5' end of the gene for the 44 kd protein. Northern RNA hybridization analysis indicates the two genes are cotranscribed into a single 3.5 kb RNA. The predicted molecular weight of the 353-residue D2 protein is 39536 and that of the 473-residue 44 kd protein is 51816. Both proteins are hydrophobic containing at least five possible membrane-spanning domains. D2 shows significant homology to the 32 kd herbicide-binding protein (Zurawski et al., (1982) Proc. Natl. Acad. Sci. USA 79, 7699-7703), and parts of the 44 kd protein show obvious similarities to parts of the 51 kd reaction-centre, chlorophyll a-binding protein of photosystem II (Morris and Herrmann (1984) Nucleic Acids Res. 12, 2837-2850). The gene for tRNASer (UGA) which is on the opposite strand to and transcribed towards the photosystem II genes is 72% homologous with the corresponding Escherichia coli tRNASer.
The translocation of the messenger RNA relative to the ribosome during peptide synthesis represents an example of a mechano-chemical reaction in which the chemical bond energy of GTP is transformed into coordinated motion. Such transformations also occur during the beating of cilia and flagellae, the contraction of muscle and the migration of chromosomes in cell division. In protein synthesis the functional geometric and energetic conditions for this transformation are well defined. For each peptide bond formed, the ribosome moves one codon along the mRNA (towards the 3' end) and one molecule of GTP is hydrolysed. Although the basic requirements of this reaction have been elucidated, the mechanism is still unresolved. We demonstrate here that translocation can be analysed as a series of binding equilibria shifted by one irreversible, GTP-consuming step. The shift in the binding equilibrium is induced by the transfer of the peptidyl moiety to the (A) site-bound aminoacyl (AA)-tRNA. This results in the A site-bound tRNA having an increased affinity for the high-affinity (P) site, and a strengthened association with the mRNA. Elongation factor (EF) G . GPT catalyses removal of the deacylated tRNA, empties the P site and at the same time loosens ribosome-mRNA association. The result of these changes is that peptidyl(PP)-tRNA . mRNA is shifted from the A site to the P site, binding of AA-tRNA . EF-Tu . GPT to the vacant A site ensuring that the process is irreversible.
The map location and nucleotide sequence of the genes and flanking regions for tRNAUUC (Glu) (trnE) and tRNAGUA (Tyr) (trnY) from spinach (Spinacia oleracea) chloroplast DNA have been determined. The genes lie approximately midway between the genes for tRNAGGU (Thr) (trnT) and tRNAGUC (Asp) (trnD) on BamH1 fragment 8b, the arrangement being trnT, 458 bp, trnE, 64 bp, trnY, 409 bp, trnD. trnE and trnY are encoded by the same DNA strand as trnD and the direction of their transcription is divergent with respect to the transcription of trnT. trnE and trnY are 89% and 74% homologous, respectively, with the corresponding Euglena gracilis chloroplast genes. The corresponding homologies between the spinach chloroplast and E. coli genes are 72% and 61%. trnE is unusual in that it has the sequence 5'ATTCNA rather than 5'GTTCNA in the TΨ arm. Northern hybridizations to chloroplast RNA with restriction fragments carrying trnE and trnY sequences indicated that both genes are transcribed in vivo.
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