Regulation of gene transcription is essential in the process of cell differentiation and development of the organism, as well as for the survival of each cell in changing environmental conditions. The importance of gene transcription and its regulation is underlined by the myriad of human disorders that result from the malfunction of the molecular components involved in this cellular process. Initiation of gene transcription in eukaryotes starts with the binding of TFIID, containing the TATA binding protein (TBP), to the DNA core promoter, and the consequent assembly of the general transcriptional machinery. Six general transcription factors and RNA Pol H constitute the minimal protein assembly for transcription, adding up to several million Daltons. TFIID is also involved in the interaction with gene-specific activators. Our ultimate goal is to understand how the eukaryotic transcription initiation machinery assembles and functions, and the structural mechanisms for gene transcription regulation. To this end we will characterize the structure of TFIID and its interaction with other general factors and with activators using cryo-electron microscopy and single-particle image reconstruction. We have recently obtained an initial structure of TFHD and its complex with full length TFILA and TFIIB at 35 A resolution by negative stain, and localized, by antibody mapping, the position of TBP within the complex. One of our immediate priorities is to obtain a 3-D reconstruction of frozen-hydrated human TFIID complexes at a resolution better than 25 A. We are interested in following the structural assembly and conformational changes of this complex machinery by generating medium resolution structures of increasingly larger complexes, with TFIID as the central core. Our final goal is to characterize the structural basis of transcription regulation by the interaction of the general transcriptional machinery with activators. To this end we will study the complexes formed by TFIID with the general activator Sp 1.