Origin of the mRNA Stoichiometry of the puf Operon...
（Received for publication，September 16，1985）
The LH-I structural genes are located 5'of the RC-L and-M structural genes on what has been designated as the puf operon of Rhodobacter sphaeroides.Analysis of puf operon expression in R.sphaeroides by Northern hybridization with probes specific for individual structural genes has identified two transcripts encoded by this operon.The large（2.6 kilobase pairs（kb））transcript contains sequences for all four polypeptides of the puf operon，whereas the small（0.5 kb）transcript，which is more abundant（10—15-fold）than the large transcript under photosynthetic growth，is homologous only to the two LH-I structural genes.Transcription of the puf operon during photosynthetic growth under saturating light conditions is increased approximately 3-fold relative to growth in the presence of oxygen while the relative ratio of these two transcripts is independent of the incident light intensity.Analysis of the turnover of the two transcripts（t1/2 of 9 and 20 min for the large and small transcripts，respectively）indicates that 5'processing is the initial step in the degradation of the large transcript and that the molar excess of the small transcript cannot be accounted for by differences in the rates of turnover of these two mRNA species.Analysis of the 5'ends of the 2.6-and 0.5-kb transcripts，their relative abundance，and stabilities indicates that these two transcripts have different 5'-ends corresponding to 75 and 104 base pairs upstream from the start of the LH-Iβ structural gene，respectively.Northern hybridization analysis with specific synthetic deoxyoligonucleotide probes confirmed that the two transcripts differ by 29 bases at their 5'-ends，suggesting that differential transcript initiation may be involved in regulating the relative levels of these two mRNA species in vivo，although we cannot rule out complex mechanisms of post-transcriptional processing.
此文发表于1986年“J.Biol.Chem.”261：10366-10374.
This work was supported by Grants GM15590 and GM31667 from the National Institutes of Health to S.Kaplan.This work was presented at the 5th International Meeting on Photosynthetic Procaryotes held in Grindelwald，Switzerland，in 1985.It was at this meeting that the nomenclature employed here for this operon was decided upon.The genus name of this bacterium has recently been changed to Rhodobacter from Rhodopseudomonas［7］.The costs of publication of this article were defrayed in par tby the payment of page charges.This article must therefore be hereby marked“advertisement”in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.
‡Predoctoral fellow supported by United States Public Health Service Training Grant GM07283 from the National Institutes of Health.(https://www.daowen.com)
In Rhodobacter sphaeroides，a purple nonsulfur photosynthetic bacterium，the two light-harvesting complexes constitute the major pigment-protein components found in the photosynthetic membrane［1］.They are composed of the light-harvesting I（B875，designated LH-I）and the light-harvesting Ⅱ（B800-850，designated LH-Ⅱ）bacteriochlorophyll-protein complexes.A third spectral complex，designated the reaction center（RC）bacteriochlorophyllprotein complex，is composed of equimolar amounts of the reaction center polypeptides L，M，and H（designated RC-L，RC-M，and RC-H）.The LH-Ⅰ complex accepts light energy from a pool of peripherally arranged LH-Ⅱ complexes（which represent the primary source of photon capture）and transfers this excitation energy to a centrally located RC complex where the primary photochemical reaction occurs［2］.The LH-Ⅰ complexes are found in the photosynthetic membrane in a fixed molar excess（approximately 12-fold）over the RC complex［3］.However，each of the two polypeptides within an LH-I complex is present in a 1∶1 stoichiometry.The LH-Ⅰ complex together with the RC complex have been termed the fixed photosynthetic unit，PUF［4］.Recently，it has been found that the structural genes for the two polypeptide subunits of LH-Ⅰ（β，α）and the RC-L and RC-M polypeptides are closely linked in a single operon in R.sphaeroides［5，6］，[2] similar to that reported for Rhodobacter capsulatus［8］，and which we designate the puf operon.The structural genes for RC-L（puf L）［6］，RC-M（puf M）［5］，and LH-Ⅰ（puf B，A）1have been sequenced，and the two LH-I genes（pu f B，A）are located proximal to the distal puf L，M genes.Since the polypeptides comprising an individual pigment-protein complex derived from the puf operon are synthesized and inserted into the photosynthetic membrane［1-3，9］in unique but different stoichiometries in both R.sphaeroides and R.capsulatus，elucidating the mechanism by which the coordinate but differential expression of these genes is accomplished of primary importance and interest.
We have previously reported10］that two transcripts are derived from the puf operon：the larger and less abundant transcript is a polycistronic mRNA possessing information for all four polypeptides；the smaller but more abundant transcript is specific for only the LH-Ⅰ β and a apoproteins.The ratio of these two transcripts reflects the ratio of the LH-Ⅰ and RC complexes found in the photosynthetic membrane.In this study we have extended our analysis of the stability，regulation by light and oxygen，and 5'-ends of the stable mRNA species encoded by the puf operon of R.sphaeroides.Similar studies were recently performed by Belasco et al.］11］for R.capsulatus，and they concluded that the large transcript，following selective degradation from the 3'-end，is the precursor of the smaller transcript.
The present work demonstrates that the two transcripts of the puf operon in R.sphaeroides have different 5'termini；the 5'terminus of the large（2.6 kb[3]）and small（0.5 kb）transcripts have been precisely mapped to 75 and 104 nucleotides，respectively，upstream of the start codon of the pufB gene.Our data also show that differences in the stability of these two transcripts may exert secondary post-transcriptional control over the stoichiometry of the LH-Ⅰ and RC complexes within the photosynthetic membrane of R.sphaeroides.Finally，we discuss these results in light of differential transcription initiation versus posttranscriptional modifications of primary transcripts as possible mechanisms for achieving the stoichiometry of the small and large puf operon transcripts.
