Binding site requirements for pea nuclear protein factor GT-1 correlate with sequences required for light-dependent transcriptional activation of the rbcS-3A gene.

Abstract

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.

Ancestral Multipartite Units in Light-Responsive Plant Promoters Have Structural Features Correlating with Specific Phototransduction Pathways

“…rbcS-CMAs 1, 2, 3, and 6; cab-CMAs 1 and 4; and the CMAs found in gapA, gapB, ppdk, atpC-D, pal, and 4-cl genes. GT-1 elements (core motif, GGTTAA; Green et al, 1988) are invariant components of only a few CMAs from dicotyledonous PhANGs such as rbcS-CMAs 2 and 3 and LsCMA1. Modules that are similar in sequence to the rbcS box I1 (GTGTGGTTAATATG), the prototype of GT-1 e l e m e n t s ( G r e e n et al, 1987), are also found in cab-CMA3, rbcS-CMA6, Pc-CMA2, rbcA-CMAl, and gapBCMAl (Fig.…”

S2F, a leaf-specific trans-acting factor, binds to a novel cis-acting element and differentially activates the RPL21 gene.

“…To investigate whether the Gill protected region was related to the GT-1 motif, we used dimers of the wild-type or mutant GT-1 binding site corresponding to box II (GGTTAA) of the pea RBCS-3A promoter (Green et al, 1988;Kuhlemeier et al,1988) as DNA competitors in gel shift assays. When an excess of unlabeled DNA containing the wild-type box II dimer sequence was included in the binding assays, all shifted bands were eliminated by competition ( Figure 4A, competitor GT-1w).…”

“…The pea RBCS-3A promoter has previously been extensively studied, and box II** has been shown by DNase I footprinting and gel retardation studies to bind the plant nuclear factor GT-1 (Green et al, 1988). However, the S2 site had not been identified in box I)** (Green et al, 1988;Gilmartin et al, 1990). To ascertain that S2F indeed binds to pea RBCS-3A box II**, we performed the following competition experiment.…”

“…The PCR product was then labeled by Y-~~P-ATP and T4 polynucleotide kinase. (3) Dimers of wild-type or mutant GT-1 binding site were prepared as described previously (Villain et al, 1994) from recombinant plasmids generously provided by N.-H. Chua (Rockefeller University, New York, NY; Green et al, 1988). (4) The 70-bp TS2w and TS2m trimer fragments were prepared by ligation of the double-stranded oligonucleotides (named S2w and S2m) containing wild-type or mutant S2 sites and were cloned into the Hindlll site of pBlueScript II KS-.…”

Structure and Light-Induced Expression of a Small Heat-Shock Protein Gene of Pharbitis nil

“…GT-1 boxes, found to be located upstream of genes encoding the small subunit of ribulose-1,5-biphosphate carboxylase, have been implicated in mediating the light-inducible expression of these genes (9,15). A six-residue core sequence, GGTTAA, within one of the GT-1 boxes has been identified as essential for the binding of GT-1 transcription factor in vitro (10). In this study, we demonstrate that expression of shsp-1 is regulated by light as well as by heat (Fig.…”