Mediator is a key regulator of eukaryotic transcription1, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II) 1-4. In the yeast S. cerevisiae, Mediator comprises 25 subunits with a total mass over 1 MDa 5,6, and is organized into three modules, termed Head, Middle/Arm and Tail 7-9. Our understanding of Mediator assembly and its role in regulating transcription has been impeded to date by limited structural information. Here, we report the crystal structure of the essential Mediator Head module (seven subunits, 223 kDa) at 4.3 Å resolution. Our structure reveals three distinct domains with the integrity of the complex centered on a bundle of ten helices from five different Head subunits. An intricate pattern of interactions within this helical bundle ensures stable assembly of the Head subunits, and provides the binding sites for general transcription factors (GTFs) and Pol II. Our structural and functional data suggest the Head module to juxtapose TFIIH and the carboxyl-terminal domain (CTD) of the largest subunit of Pol II, thereby facilitating CTD phosphorylation. Our results reveal architectural principles underlying the role of Mediator in the regulation of gene expression.

Summary SecM is an E. coli secretion monitor capable of stalling translation on the prokaryotic ribosome without co-factors. Biochemical and structural studies have demonstrated that the SecM nascent chain interacts with the 50S subunit exit tunnel to inhibit peptide bond formation. However, the timescales and pathways of stalling on a mRNA remain undefined. To provide a dynamic mechanism for stalling, we directly tracked the dynamics of elongation on ribosomes translating the SecM stall sequence (FSTPVWISQAQGIRAGP) using single-molecule fluorescence techniques. Within one minute, three peptide-ribosome interactions work cooperatively over the last 5 codons of the SecM sequence, leading to severely impaired elongation rates beginning from the terminal proline and lasting 4 codons. Our results suggest that stalling is tightly linked to the dynamics of elongation and underscore the roles that the exit tunnel and nascent chain play in controlling fundamental steps in translation.

SUMMARY The traditional view of macrolide antibiotics as plugs inside the ribosomal nascent peptide exit tunnel (NPET) has lately been challenged in favor of a more complex, heterogeneous mechanism, where drug-peptide interactions determine the fate of a translating ribosome. To investigate these highly dynamic processes, we applied single-molecule tracking of elongating ribosomes during inhibition of elongation by erythromycin of several nascent chains, including ErmCL and H-NS, which were shown to be respectively sensitive and resistant to erythromycin. Peptide sequence-specific changes were observed in translation elongation dynamics in the presence of a macrolide-obstructed NPET. Elongation rates were not severely inhibited in general by the presence of the drug; instead, stalls or pauses were observed as abrupt events. The dynamic pathways of nascent-chain dependent elongation pausing in the presence of macrolides determine the fate of the translating ribosome - stalling or read-through.

The recent growth in single molecule studies of translation has provided an insight into the molecular mechanism of ribosomal function. Single molecule fluorescence approaches allowed direct observation of the structural rearrangements occurring during translation and revealed dynamic motions of the ribosome and its ligands. These studies demonstrated how ligand binding affects dynamics of the ribosome, and the role of the conformational sampling in large-scale rearrangements intrinsic to translation elongation. The application of time-resolved cryo-electron microscopy revealed new conformational intermediates during back-translocation providing an insight into ribosomal dynamics from an alternative perspective. Recent developments permitted examination of conformational and compositional dynamics of the ribosome in real-time through multiple cycles of elongation at the single molecule level. The zero-mode waveguide approach allowed direct observation of the compositional dynamics of tRNA occupancy on the elongating ribosome. The emergence of single molecule in vivo techniques provided insights into the mechanism and regulation of translation at the organismal level.

A high-throughput assay for real-time measurement of translation rates in cell-free protein synthesis (SNAP assay) is described. The SNAP assay enables quantitative, real-time measurement of overall translation rates in vitro via the synthesis of O 6 -alkylguanine DNA O 6 -alkyltransferase (SNAP). SNAP production is continuously detected by fluorescence produced by the reaction of SNAP with a range of quenched fluorogenic substrates. The capabilities of the assay are exemplified by measurements of the activities of Escherichia coli MRE600 ribosomes and fluorescently labeled E. coli mutant ribosomes in the PURExpress translation system and by determination of the 50% inhibitory concentrations (IC 50 ) of three common macrolide antibiotics.