Rubisco Promoters

Fig. 3.6 Human serum albumin (HSA) was produced during the sprouting of transgenic rape-seeds. A light-inducible Rubisco promoter was used to control transgene expression. The highest yield was obtained with light induction after three days of continuous darkness. Sprouting was carried out in an airlift fermentor at room temperature. The sprouting medium was water.

P Synechococcus sp.PCC7942 RuBisCO promoter T Synechococcus sp.PCC7942 RuBisCO terminator icfA Synechococcus sp.PCC7942 CA gene... 

Rubisco exists in three forms an inactive form designated E a carbamylated, but inactive, form designated EC and an active form, ECM, which is carbamylated and has Mg at its active sites as well. Carbamylation of rubisco takes place by addition of COg to its Lys ° e-NHg groups (to give e—NH—COO derivatives). The COg molecules used to carbamylate Lys residues do not become substrates. The carbamylation reaction is promoted by slightly alkaline pH (pH 8). Carbamylation of rubisco completes the formation of a binding site for the Mg that participates in the catalytic reaction. Once Mg binds to EC, rubisco achieves its active CM form. Activated rubisco displays a Ai, for CO2 of 10 to 20... 

Substrate RuBP binds much more tightly to the inactive E form of rubisco (An = 20 nM) than to the active ECM form (A, for RuBP = 20 ixM). Thus, RuBP is also a potent inhibitor of rubisco activity. Release of RuBP from the active site of rubisco is mediated by rubisco activase. Rubisco activase is a regulatory protein it binds to A-form rubisco and, in an ATP-dependent reaction, promotes the release of RuBP. Rubisco then becomes activated by carbamylation and Mg binding. Rubisco activase itself is activated in an indirect manner by light. Thus, light is the ultimate activator of rubisco. 

Transcriptional inhibitors could be used simultaneously. Rifampicin blocks chloroplast and mitocondrian RNA synthesis [23, 24], while tagetitoxin is a very specific inhibitor of chloroplast RNA polymerase [25]. Treatment with these antibiotics does not inhibit Rubisco SSU synthesis since the promoter is part of the nuclear genome, while the cytosolic ribosomes are not affected by streptomycin. Therefore SSU promoters can be used to drive transgene expression and facilitate the accumulation of recombinant proteins. Expressed proteins are targeted to a suitable cellular compartment, such as the cytoplasm, apoplastic space or chloroplast, depending on the nature of the protein. 

We have isolated Rubisco small subunit promoters from several plant species and tested their strength with gusA and ALB (human serum albumin) transgenes in sprouts. The highest level of expression in Brassica napus sprouts has been obtained... 

Human hemoglobin (< + P) CaMVenhanced 35S promoter/ CaMV 35S terminator Transit peptide of small subunit of RubisCO of Pisum sativum N. tabacum 0.05% of seed protein 37... 

Before substrate binding can take place, rubisco must first be activated. This occurs via carbamylation (reaction with CO2) of an essential Lys residue . This promotes the binding of an essential Mg + ion after which the active site is complete. Rubisco can now recognize and bind the first substrate which is ribulose-P2 (D-ribulose 1,5-bisphosphate) . The substrate is bound to the Mg + ion via an inner-sphere coordination of the C2-carbony 1 and C3-hydroxyl groups which appropriately positions and activates the ribulose-P2 for subsequent reaction. Substrate binding causes a flexible loop to close over the active site which buries the active site deep within the protein and restricts access to a small channel just large enough for CO2 (and 02) . 

Carbon dioxide fixation is catalyzed by the enzyme ribulosebisphosphatecar-boxylase oxygenase (RuBisCO), which is the most abundant natural enzyme worldwide, and the least selective. In fact, RuBisCO does not follow the usual selectivity of enzymes, and at the same time promotes the carboxylation of ribulose (a C-5 sugar) to afford a C-6 sugar (with carbon fixation) and the oxidation of the same C-5 sugar, with a selectivity close to 50% [2]. 

Plasmids containing the cyanobacterial CA gene icfk) [3], promoter and terminator derived from RuBisCO and the ampicillin resistant gene were constructed (Fig.2). The resulting plasmids were introduced into Synechococcus sp. PCC7942 [4]. 

The In Vitro Reconstitution Of Active Rubisco. To explore the molecular mechanism by which Rubisco assembly is promoted by the chaperonins, an in vitro system was developed. We chose the dimeric, simplifi form of the Rhodospirilum nibrum Rubisco as our substrate, to be denatured with either 8M urea, 6M guanidium-HCl or acid treatment The E, coli chaperonins, now referred to as cpn60 ( EL) and cpnlO (groES) were purified to homogeneity (22),... 

A further aspect of molecular synthesis in a high-temperature environment is the recognition of "heat-shock proteins," proteins whose synthesis is promoted by heat stress. One particular group of heat-shock proteins is seen as particularly relevant to the development of photosynthesis. These are the chaperonins, which play an important role in binding and shaping the enzyme rubisco. 

Figure 8. Strong light induced GUS-reporter eaq>ression is driven from a new SRSlpt soybean rubisco smaU subunit vector. (A) The SRSlpt/GUS fiaion construct was transformed into Arabidopsis and one of several stable transgenic lines is shown after staining for GVS eqtression. The indigo blue product of GUS activity was developed by incubating the plant in the colorless x-gluc substrate, after 24 hours offixation and bleaching of the tissue in ethanol. The GUS reporter was fused between the SRSl promoter and terminator to test speciftcity of the new SRSlpt vector. (B) A wild-type Arabidopsis plant is similarly stained for GUS activity, but shorn no color.

COg concentrations may result in a higher proportion of the enzyme being in the enediol form (21), also promoting slow inactivation (Fig. 4b). Both of these effects may be significant in regulating Rubisco s activity in vivo. 

The results of this paper indicate that L subvmits of higher plant Rubisco have a high tendency to form incorrect self aggregations and this tendency was reduced by the presence of BP. I suggest that the function of BP is to act as a supporting column where newly synthesized L subunits bind to and this binding promotes the correct folding and association of the L subunits. 

Ribose 5-phosphate is a negative effector, while FBP was observed to be a very potent inhibitor of rubisco activation at low CO2 concentrations even though it eventually acts as a good positive effector (11). As shown in Fig 5, activation was fairly rapid in the presence of PGA and rubisco activase addition had minimal effect on the kinetics. In contrast, rubisco activase greatly increased the rate and extent of activation of rubisco in the presence of FBP and ribose 5-phosphate. These results provide further support for a scheme in which rubisco activase, by alleviating the inhibitory effects of FBP, ribose 5-phosphate and RuBP on the forward spontaneous activation process, but not their inhibitory effects on the backward deactivation process, promotes the rapid attainment of a higher steady state activation level... 

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