Plant Molecular Biology 33: 635–640, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
RNA processing alters open reading frame stoichiometry from the large ATP
synthase gene cluster of spinach chloroplasts
Tracy Hotchkiss and Margaret J. Hollingsworth
Department of Biological Sciences, State University of New York, Buffalo, NY 14260, USA (
Received 23 April 1996; accepted in revised form 4 November 1996
Key words: ATP synthase complex, chloroplast gene expression, post-transcriptional regulation, RNA processing
The largeATP synthase gene cluster of spinach chloroplasts is a multigenic cluster that encodes the small ribosomal
subunit 2 followed by four ATP synthase subunits. The stoichiometry of the ATP synthase gene products from
this cluster changes markedly between transcription and assembly of the complex. The two primary transcripts
from this gene cluster undergo a complex series of RNA processing steps. Here we show that the extensive RNA
processing that the large ATP synthase gene cluster transcripts undergo results in a substantial change in the
stoichiometryof complete open reading frames (ORFs) of the four ATP synthase genes. Processing directly affects
the stoichiometry of open reading frames from this gene cluster by intragenic cleavage. It may also affect open
reading frame stoichiometry more indirectly, but equally signiﬁcantly, by cleavage-induced alteration of stability
of some of the processed transcripts relative to the others.
All of the polypeptidecomplexes embedded in chloro-
plast thylakoid membranes are derived from a com-
bination of plastid and nuclear-encoded subunits .
Efﬁcient biogenesis of chloroplast complexes requires
precise coordination of chloroplast and nuclear gene
expression to produce polypeptides in the correct stoi-
chiometries [12, 19, 21, 23, 27].
Mechanisms by which plant cells coordinately
beenintensely studied[12,19, 21, 23, 27]. The expres-
sion of nuclear-encoded chloroplast genes is primarily
. In contrast, transcription of most chloroplast-
encoded genes is essentially constitutive, with glob-
al RNA ﬂuctuations depending on the developmental
stage of the chloroplast [5, 11, 12, 13, 23]. Post-
transcriptional regulation provides fundamental con-
trol for most plastid gene expression.
Many chloroplast-encoded genes are arranged in
multigenic clusters reminiscent of bacterial genomes.
These clusters often encode genes required for more
than one chloroplast complex [13, 21, 25, 27, 31, 32].
Our research concentrates on the large ATP synthase
gene cluster from spinach chloroplasts. This cluster
encodes (in order) the small ribosomal subunit pro-
tein 2 (rps2), and four ATP synthase subunits: CF
-III (atpH), CF
(Fig. 1) ( for nomenclature ). The transcripts
of this cluster display a complex pattern of RNA mat-
uration [18, 34]. The two primary transcripts origin-
ate upstream of rps2 or atpH and terminate down-
stream of atpA. This results in an initial RNA ratio
of X:1:1:1 for the four ATP synthase gene products
(atpI:atpH:atpF:atpA), with the value of X depending
on the relative strengths of the two promoters .
After processing, there are more than thirty RNA spe-
cies, all of which appear to be bound by ribosomes
(N. Stollar and M.J. Hollingsworth, unpublished).The
polypeptides encoded by the large ATP synthase gene
complex [22, 36]. No combination of transcription
from the two promoters could yield a ratio of RNA for