THE CONTRIBUTION OF AAUAAA AND THE UPSTREAM ELEMENT UUUGUA TO THE EFFICIENCY OF MESSENGER-RNA 3'-END FORMATION IN PLANTS

The requirement for sequence specificity in the AAUAAA motif of the ca uliflower mosaic virus (CaMV) polyadenylation signal was examined by s aturation mutagenesis. While deletion of AAUAAA almost abolished proce ssing at the CaMV polyadenylation site, none of the 18 possible single base mutations had a dramatic effect on processing efficiency. The ef fect of replacing all six nucleotides simultaneously varied depending on the sequence used, but some replacements were as detrimental as the deletion mutant. Taken together, these results confirm that AAUAAA is an essential component of the CaMV polyadenylation signal, but indica te that a high degree of sequence variation can be tolerated. A repeat ed UUUGUA moth was identified as an important upstream accessory eleme nt of the CaMV polyadenylation signal. This sequence was able to induc e processing at a heterologous polyadenylation site in a sequence-spec ific and additive manner. The effect of altering the spacing between t his upstream element and the AAUAAA was examined; moving these two ele ments closer together or further apart reduces the processing efficien cy. The upstream element does not function to signal processing at the CaMV polyadenylation site if placed downstream of the cleavage site. Analysis of further upstream sequences revealed that almost all of the 200 nt fragment required for maximal processing contributes positivel y to processing efficiency. Furthermore, isolated far upstream sequenc es distinct from UUUGUA were also able to induce processing at a heter ologous polyadenylation site.