Binding site requirements for pea nuclear protein factor GT-1 correlate with sequences required for light-dependent...

Nuclear protein factor GT-1 binds to sequence boxes II, III, II* and III* upstream of the light-responsive pea rbcS-3A gene. We have shown previously that box II and box III are required for expression of rbcS-3A when redundant elements upstream of -170 (relative to the transcription start site) are removed. Here we present evidence that deletion and substitution mutations downstream of -170 which eliminate expression also decrease binding. Using a series of 2 bp substitution mutations we have defined a core of six residues (GGTTAA) within box II (GTGTGGTTAATATG) that are critical for binding. The most detrimental mutation for binding, which changes the double Gs to Cs, is sufficient to eliminate detectable expression in vivo when only 170 bp of 5' flanking sequences are present. The simplest interpretation of these data is that GT-1 is an activator of rbcS-3A transcription. Footprinting experiments show that GT-1 from both light-grown and dark-adapted plants binds to the same sequences in vitro. Therefore, the lack of expression of rbcS-3A in the dark is not due to the absence of GT-1. In our analysis of the sequence elements upstream of -170, we have mapped two additional GT-1 sites (boxes II** and III**) between -330 and -410. The similarities and differences among the GT-1 sites located upstream and downstream of -170 are discussed in terms of the different sequence requirements for rbcS-3A expression during development.