Cis-acting Elements and Trans-acting Factors

Tissue-Specific Binding Of Trans-Acting Factors

Tissue-Specific Binding Of Trans-Acting Factors

The question of how a gene is expressed differentially is key to our understanding of genetic regulation. One manner to study this question is to map the functional sequence domains of a gene and determine what sequences are bound by proteins (presumably trans-acting factors) during expression in different tissues. This approach was utilized to study the expressiion of the rbcS gene family of tomato.

The five members of the rbcS gene family are rbcS1, rbcS2, rbcS3A, rbcS3B, and rbcS3C. As you would expect of a gene involved in photosynthesis, the highest level of expression of the gene family is in the leaf [PNAS (1987) 84:7104, Table 1]. Another tissue in which the gene is expressed is in the green, immature tomato fruit. Analysis of the expression of each individual family member determined that only rbcS1 and rbcS2 were expressed in that tissue (Table 2). rbcS is also expressed in etiolated tissue, and the bulk of this expression is accounted for by the activity of the rbcS1 and rbcS3 genes (Table 2). Gene expression is of the rbcS gene family is turned off in non-photosynthetic tissues.

The different promoter sequences of each member of the rbcS gene family were analyzed by gel retardation assays [Plant Cell (1991) 3:1305]. These assays defined 21 different sequence motifs. Not all of the motifs were found in each promoter, though. Next, nuclear protein extracts were isolated from cotyledon, leaf, young fruit, and mature fruit and root tissue, and it was determined to which sequences nuclear proteins bound to with each rbcS gene family member promoter (Figure 6). The G-box motif was the only sequence to which a protein bound in non- photosynthetic tissues. Variation in nuclear protein binding patterns was noted among extracts from different tissue sources. These results suggest that the binding of nuclear proteins can account for the difference in gene expression patterns of the different members of the rbcS gene family.

The following table shows the promoter sequences found in various rbcS promoters, and the location where DNA-binding proteins that bind the various sequences are expressed.

Conserved rbcS Motif Present in promoter of: DNA binding proteins expressed in:

L-box rbcS1, 3A Leaf, cotyledon

Motif 2 (related sequences are I Box and GATA motif) rbcS1, 2, 3A Leaf, cotyledon binding to rbcS1 and 2

G-Box (Motif 3) rbcS1, 2, 3A All tissues except cotyledons; these mRNAs are not present in roots

Light regulatory sequences (motif 18) rbcS1, 3A, 3B, 3C All except root; these genes are not expressed in roots

TATA Box rbcS1, 3A, 3B, 3C All except root; these genes are not expressed in roots

A/T and G/C rich regions rbcS1, 3A, 3B, 3C All except root

G/C rich region rbcS1 All except root