David M. Chao

A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast

We report genetic and biochemical evidence that the RNA polymerase II carboxy-terminal domain (CTD) interacts with a large multisubunit complex that contains TATA-binding protein (TBP) and is an integral part of the transcription initiation complex. The isolation and characterization of extragenic suppressors of S. cerevisiae RNA polymerase II CTD truncation mutations led us to identify SRB2, SRB4, SRB5, and SRB6 as genes involved in CTD function in vivo. SRB2 was previously isolated and shown to encode a 23 kd TBP-binding protein. The four SRB proteins and a portion of cellular TBP are components of a high molecular weight multisubunit complex that is tightly bound to RNA polymerase II. This SRB-TBP complex binds specifically to recombinant CTD protein. In vitro transcription and template commitment assays confirm that SRB2 and SRB5 are components of a functional preinitiation complex and are required for efficient transcription initiation.

RNA Polymerase II Holoenzyme Contains SWI/SNF Regulators Involved in Chromatin Remodeling

The RNA polymerase II holoenzyme contains RNA polymerase II, a subset of general transcription factors and SRB regulatory proteins. We report here that SWI and SNF gene products, previously identified as global gene regulators whose functions include remodeling chromatin, are also integral components of the yeast RNA polymerase II holoenzyme. The SWI/SNF proteins are components of the SRB complex, also known as the mediator, which is tightly associated with the RNA polymerase II C-terminal repeat domain. The SWI/SNF components provide the holoenzyme with the capacity to disrupt nucleosomal DNA and thus facilitate stable binding of various components of the transcription initiation complex at promoters.

Immersion zone-plate-array lithography

An immersion scheme is used to improve resolution, exposure latitude, and depth-of-focus in zone-plate-array lithography (ZPAL). We believe this is the first implementation of an immersion scheme in a maskless lithography system. Replacing air with de-ionized water as the medium between the zone-plate array and the substrate effectively increases the system’s numerical aperture and consequently, enhances its patterning capabilities. The design and fabrication process of an immersion zone plate is described. Its behavior is then characterized through the experimental reconstruction of its point-spread function, and compared to the theoretical model. A wide variety of patterns were printed, demonstrating the improved lithographic performance of immersion ZPAL.

Purification of yeast RNA polymerase II holoenzymes.

Publisher Summary This chapter describes methods for the purification and assay of the two largest forms of RNA polymerase II holoenzyme from the yeast Saccharomyces cerevisiae . Use of the purified holoenzyme for transcription studies in vitro should yield new and exciting clues regarding the mechanisms underlying transcriptional regulation. RNA polymerase II can be purified from yeast in a high molecular weight complex called an RNA polymerase II holoenzyme. This form of RNA polymerase II appears to be responsible for transcription initiation in vivo . All forms of RNA polymerase II holoenzyme described thus far lack the general transcription factors TBP and TFIIE (transcription factor IIE), and these factors must be added to obtain transcription in vitro. TFIIB is also required for transcription by some holoenzyme preparations. Holoenzyme and factor preparations should be dialyzed against a low-salt buffer, such as buffer F, to reduce the salt content prior to assay. Alternatively, the salt concentration can be determined by conductivity measurements, and the salt concentration in the transcription assay buffer can be adjusted accordingly.

Octave-spanning supercontinuum generation for an Er-doped fiber laser frequency comb at a 1 GHz repetition rate

We developed a 1 GHz Er-doped femtosecond fiber laser system providing 2nJ pulses at ∼100fs durations and demonstrated octave-spanning supercontinuum generation from 1µm – 2.4µm that is suitable for 1f–2f stabilizing the frequency comb.

Maskless Lithography Using Diffractive-Optical Arrays

An optical maskless-lithography system is described that is capable of patterning complex geometries at very high resolution, and in a cost-effective manner with potential applications in the fabrication of nanostructures for photonic devices.

Self-referenced Erbium fiber laser frequency comb at a GHz repetition rate

An Erbium fiber laser frequency comb operating at a GHz repetition rate is reported. The f_ceo and f_rep stabilized all-fiber system produces 2nJ sub-100fs pulses to seed octave-spanning supercontinuum generation spanning 1 μm-2.4 μm.

Femtosecond sources for optical arbitrary waveform generation

Advances in high repetition-rate femtosecond laser technology for optical arbitrary waveform generation will be described. Combs spanning two octaves, from 500nm to 2μm, based on GHz modelocked Ti:sapphire and erbium-fiber lasers, have been carrier-envelope stabilized and frequency referenced.

Enabling nanoscale science and engineering via highly flexible, low-cost, maskless lithography

The role of lithography in the future of nanoscale science and engineering is to put high-density spatial information into nanoscale assemblies. Because information content determines the functionality of such assemblies, lithography will be a key enabler. Conventional lithographic techniques generally lack the flexibility, low cost and the resolution that research in nanoscale science and engineering requires. Although no single lithographic technique is likely to be a panacea, it is important to seek novel approaches that meet the needs of researchers, and open a path to directly manipulating nanoparticles and macromolecules. We review the various forms of lithography and focus special attention on maskless zone-plate-array lithography, assessing its impact, advantages and extendibility to the limits of the lithographic process. Nanoscale assemblies will require control at the macromolecular level, and this has begun with research on templated self assembly. Going beyond that to the control and utilization of the information content of nanoparticles and molecules will require innovations whose origin is uncertain at this point.