Study of the binding of RNA polymerase by a recombinant plasmid using electron microscopy

RNA-polymerase of E. coli was bound in vitro under physiological conditions to a recombinant plasmid pBR322 carrying two identical segments of bacteriophage T4 DNA, each containing a complete gene coding for T4 DNA ligase. After fixation of the complex with formaldehyde it was analysed by electron microscopy. A map of binding sites of the enzyme to DNA was obtained after a statistical assessment of micrographs. The inserted repeat revealed itself in the map as two regions of identical binding patterns, thus proving the adequacy of the preparation procedure. In pBR322 the strong binding sites correlate with the position of promoters. Also, apart from this, there are other strong binding sites within the T4 sequences which correlate strongly with the regions of abnormally high AT content. That means that under physiological conditions the RNA polymerase forms strong binding complexes with any AT rich DNA regions, as well as with real promoters.     

Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron microscopy

The structure of the multisubunit yeast DNA polymerase epsilon (Pol epsilon) was determined to 20-A resolution using cryo-EM and single-particle image analysis. A globular domain comprising the catalytic Pol2 subunit is flexibly connected to an extended structure formed by subunits Dpb2, Dpb3 and Dpb4. Consistent with the reported involvement of the latter in interaction with nucleic acids, the Dpb portion of the structure directly faces a single cleft in the Pol2 subunit that seems wide enough to accommodate double-stranded DNA. Primer-extension experiments reveal that Pol epsilon processivity requires a minimum length of primer-template duplex that corresponds to the dimensions of the extended Dpb structure. Together, these observations suggest a mechanism for interaction of Pol epsilon with DNA that might explain how the structure of the enzyme contributes to its intrinsic processivity.     

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy

The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.     

The ribosome-structure and functional ligand-binding experiments using cryo-electron microscopy

Cryo-electron microscopy has greatly advanced our understanding of the basic steps of protein synthesis in the bacterial ribosome. This article gives an overview of what has been achieved so far. Through three-dimensional visualization of complexes that represent the ribosome in defined binding states, locations were derived for the tRNA in A, P, and E sites, as well as the elongation factors. In addition, the pathways of messenger RNA and the exiting polypeptide chain could be inferred.