ABSTRACT
The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.
Subject(s)
RNA Polymerase I/analysis , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae/enzymology , Cryoelectron Microscopy , Image Processing, Computer-Assisted , Models, Molecular , Negative Staining , Protein Conformation , Protein Interaction Mapping , Protein Subunits , RNA Polymerase I/ultrastructure , RNA Polymerase II/chemistry , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/ultrastructure , Sequence DeletionABSTRACT
The transcription factor TFIID is a large multiprotein complex, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), which plays a key role in the regulation of gene expression by RNA polymerase II. The three-dimensional structure of yeast (y) TFIID, determined at approximately 3 nm resolution by electron microscopy and image analysis, resembles a molecular clamp formed by three major lobes connected by thin linking domains. The yTFIID is structurally similar to the human factor although the clamp appears more closed in the yeast complex, probably reflecting the conformational flexibility of the structure. Immunolabelling experiments showed that nine TAFs that contain the histone fold structural motif were located in three distinct substructures of TFIID. The distribution of these TAFs showed that the previously reported pair-wise interactions between histone fold domain (HFD)-containing TAFs are likely to occur in the native yTFIID complex. Most of the HFD-containing TAFs have been found in two distinct lobes, thus revealing an unexpected and novel molecular organization of TFIID.
Subject(s)
Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Transcription Factors, TFII/chemistry , Transcription Factors/chemistry , Amino Acid Motifs , Animals , Dimerization , Drosophila Proteins/chemistry , Histones/chemistry , Humans , Image Processing, Computer-Assisted , Macromolecular Substances , Microscopy, Electron , Microscopy, Immunoelectron , Models, Molecular , Multiprotein Complexes , Precipitin Tests , Protein Conformation , Protein Interaction Mapping , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/ultrastructure , Species Specificity , Structure-Activity Relationship , Transcription Factor TFIID , Transcription Factors/classification , Transcription Factors/ultrastructure , Transcription Factors, TFII/ultrastructureABSTRACT
Two distinct proteins, streptavidin and HupR, bind and form regular helical arrays on the surface of multiwalled carbon nanotubes under appropriate conditions (see picture). The decoration of the outer surface of these nanostructures with biological macromolecules was investigated by electron microscopy.
