Abstract
Higher plant plastid genomes encode rRNAs, tRNAs, and protein subunits of the RNA polymerase, ribosomes, and the photosynthetic apparatus which vary over 1000-fold in abundance. Quantitative analysis of transcription and RNA levels was carried out on 15 plastid genes which are located in 14 different transcription units covering 50% of the barley plastid genome. Transcription of 16S rRNA, trnfM-trnG, and trnK was high relative to most other plastid genes. Transcription of trnfM-trnG was 5 times greater than trnK indicating that differences in tRNA levels in plastids could be due, in part, to differences in transcription. Among the protein coding genes, mRNA levels varied over 900-fold and transcription over 300-fold. The gene showing the lowest transcription rate and mRNA level, rpoB, is located in a gene cluster which encodes subunits of the plastid RNA polymerase (rpoB-rpoC1-rpoC2). RpoA, which encodes the alpha subunit of the RNA polymerase, was located in a gene cluster encoding ribosomal proteins (rpl23, rps19, rpl16) and infA. RNA from this gene cluster is 30-fold more abundant than rpoB mRNA, suggesting that expression of rpoA is regulated at the level of translation or protein stability. Polycistronic operons encoding subunits of the photosynthetic apparatus (psbB-psbH-petB-petD; psbK-psbI-psbD-psbC; atpB-atpE; psaA-psaB) had higher transcription rates and correspondingly higher mRNA levels than genes which encode ribosomal proteins or RNA polymerase subunits. RbcL and psbA, which are located in separate transcription units, exhibited the highest transcription rates and mRNA levels. Correspondence between transcription rate, mRNA level, and protein abundance indicates that transcription is a primary determinant of barley plastid gene expression. In addition, a 30-fold variation in predicted mRNA stability was observed which further increases the dynamic range of plastid mRNA abundance.
Highlights
Higher plant plastidgenomes encode rRNAs,tRNAs, kbp,’ dependingonplant species (for review, see Palmer and proteinsubunits of the RNA polymerase,ribo- (1990))
Among the protein coding genes, mRNA levels are located within tRNAgene clusters, two are located within varied over 900-fold and transcription ove3r00-fold. the rrn transcription unit, andsome are located within or at
Plastid and nuclear genes encoding subtranscription is a primary determinant of barley plas- units of a common protein complex need to be expressed a t tid gene expression.In addition,a 30-fold variation in similar levels, while genesencoding subunits of different predicted mRNA stability was observed which furthercomplexes are often needed at very different levels
Summary
The organization of genes encoding proteins with related functions into transcription units may facilitate the coordinated production of subunits of a single protein complex and/or allow groups of related genes to be blottingandPCR amplification reactions.Probesfor rpoA, rpoB, rp116, andndhA were generated by PCR amplification (Table I). GAT-3', a 2.5-kbp PCR product between the inverse complement of intooperons may facilitatecoordinated expression, many the rpsl primer and rp116 primer 5'-CATTCTTCCTCTATG-3', a plastid operons arecomprised of multiple transcription units (i.e. psbK-psbI-psbD-psbC-orf; atpB-atpE)(Sextonet al., 1990b; Bennett et aL, 1990) and extensive RNAprocessing of primary transcripts is often observed(Westhoff and Herrmann, 1988; Tanaka et al, 1987). Plastid genes a t one stage of chloroplast development in RNA was concentrated by centrifuging 15 min in a microcentrifuge, barley This analysirsevealed over 300-fold variation in tran- washed with 70% ethanol, and dried 2 min in a speed vac. RNA was resuspended in 50-100 pI of T E and residual CsCl removed usinga Sephadex G-50-80 spin column.RNA was separatedon
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