On the use of the distal promoter in the zein genetic system

Zein is abundantly synthesized in the developing endosperm. The exact mechanism leading to the intense synthesis is unknown although the multigenic character of the system, the promoter set up and efficient translation of zein mRNAs may all contribute to this feature.

In this context our recent finding of multiple promoters before zein genes (see MNL 58:88-89, 1984) may be of relevance, in particular since the observed differences in the various promoter structures may allow a regulatory influence on efficiency of gene expression. However, for this to occur the RNAs synthesized from the P1 promoter regions lying 1 kb upstream from the start of protein synthesis must finally lead to protein synthesis.

We now have evidence which shows that this is indeed the case. Transient transformation experiments performed with cloned zein genes in the alga Acetabularia (system described in MNL 59:48, 1985) show that zein protein is synthesized in this system from RNA derived from the P1 region. Mutants with an impaired P1 structure and otherwise intact 5' flanking regions do not lead to zein synthesis, indicating that the P1 structure does generate RNAs with translational capacity and that P2 is not active in this system.

The expression of the 700-800 bp long coding region from a 1.6-1.8 kb long RNA is apparently preceded by a splicing step which transfers the 5' end of the RNA (presumably containing a cap structure) in front of the gene:

Evidence in support of such a mechanism was obtained by a combination of S1 mapping and primer extension experiments in the region of P2, and by DNA sequence comparison. The sequence of the relevant regions does show intron border sequences, branchpoint sequences and partial homology to snRNA at the expected positions.

The proposed mechanism is appealing since it accommodates the use of the cap structure of the primary transcript and also avoids the interference of the more than 15 AUG codons present in the 1 kb flanking sequences.

Figure.

J. Brown, Chr. Wandelt and G. Feix
 
 


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