In situ transcription and splicing in the Balbiani ring 3 gene

The Balbiani ring 3 (BR3) gene contains 38 introns, and more than half of t hem are co-transcriptionally excised. We have determined the in situ struct ure of the active BR3 gene by electron tomography, Each of the 20-25 nascen t transcripts on the gene is present together with splicing factors and the RNA polymerase II in a nascent transcript and splicing complex, here calle d the NTS complex. The results indicate that extensive changes in overall s hape, substructure and molecular mass take place repeatedly within an NTS c omplex as it moves along the gene. The volume and calculated mass of the NT S complexes show that, maximally, one complete spliceosome is assembled on the multi-intron transcript at any given time point, The structural data sh ow that the spliceosome is not a structurally well-defined unit in situ and that the C-terminal domain of the elongating RNA polymerase II cannot carr y spliceosomal components for all introns in the BR3 transcript. Our data i ndicate that spliceosomal factors are continuously added to and released fr om the NTS complexes during transcription elongation.