Homology between alpha 2(V) and alpha 1(III) collagen promoters and evidence for negatively acting elements in the alpha 2(V) first intron and 5' flanking sequences.

We have isolated a 17 kilobase pair (kb) genomic clone containing the 5' portion of the human alpha 2(V) collagen gene. Nucleotide sequence was determined for 1671 base pairs (bp) comprising the promoter region, first exon and 334 bp of the first intron, and the major transcriptional start site determined by primer extension and S1 nuclease analysis. Sequence comparison revealed the alpha 2(V) promoter to be similar in structure to the promoter of the alpha 1(III) collagen gene. This is the first instance of such similarities between promoter regions of genes encoding different fibrillar collagen chains. Homology, in 5' flanking sequences, extends upstream to about nucleotide -120 in each gene and is particularly striking near the TATTTA sequence (TATA box) present in each promoter. Some homology also surrounds the two transcription start sites. The 5' untranslated regions of the two genes also show strong homology. Chimeric chloramphenicol acetyltransferase (CAT) constructs were prepared with various fragments from the 5' portion of the alpha 2(V) gene. Transient expression assays, in human fibroblasts, localized the functional alpha 2(V) promoter to the region of 5' flanking sequence conserved between the alpha 2(V) and alpha 1(III) genes. Expression assays also identified negatively acting elements, in intron and 5' flanking sequences, which inhibit transcription from the alpha 2(V) promoter.