We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis.

We have used UV-laser mediated cross-linking, DNase I footprinting and KMnO4 reactivity to probe the interaction between T7 RNA polymerase (RNAP) and a consensus promoter during the early stages of transcription. In a binary complex formed in the absence of substrate on a supercoiled plasmid, direct contacts were observed on the template (T) strand at positions -17, -5, and +3 and on the nontemplate (NT) strand at position -8. These contacts lie within the DNase I cleavage footprint from positions -21 to +11 on the T strand and from positions -17 to +16 on the NT strand and straddle sites of enhanced reactivity of thymines to KMnO4 at position -3 on the T strand and position -2 on the NT strand. Use of supercoiled plasmid templates has allowed the mapping of contacts in the initiation region of the promoter in the binary complex for the first time. Upon addition of GTP, T7 RNAP enters a reiterative mode of synthesis, producing a ladder of poly(G) products. Under these conditions the downstream contact on the T strand switched from position +3 to +4 and +5 while the contact at position -17 was maintained. Under conditions in which the synthesis of transcription products is limited to 6-7 nucleotides, only the contact at position -17 on the T strand was preserved. A comparison of these results with the interaction of Escherichia coli RNA polymerase at the lac promoter reveals strong similarities in the manner in which these polymerases recognize their promoters.