Engineering topics

Bailey, Scott

Publications and source records attributed to Bailey, Scott.

5 records

DOE OSTI2016

Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation

The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo. Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multi-subunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Structural basis for promiscuous PAM recognition in type I–E Cascade from E. coli

Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. Furthermore, these observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2015

Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli

In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli, short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro. Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2011

On the Importance of the Flare's Late Phase for the Solar Extreme Ultraviolet Irradiance

The new solar extreme ultraviolet (EUV) irradiance observations from NASA Solar Dynamics Observatory (SDO) have revealed a new class of solar flares that are referred to as late phase flares. These flares are characterized by the hot 2-5 MK coronal emissions (e.g., Fe XVI 33.5 nm) showing large secondary peaks that appear many minutes to hours after an eruptive flare event. In contrast, the cool 0.7-1.5 MK coronal emissions (e.g., Fe IX 17.1 nm) usually dim immediately after the flare onset and do not recover until after the delayed second peak of the hot coronal emissions. We refer to this period of 1-5 hours after the fl amrea sin phase as the late phase, and this late phase is uniquely different than long duration flares associated with 2-ribbon flares or large filament eruptions. Our analysis of the late phase flare events indicates that the late phase involves hot coronal loops near the flaring region, not directly related to the original flaring loop system but rather with the higher post-eruption fields. Another finding is that space weather applications concerning Earth s ionosphere and thermosphere need to consider these late phase flares because they can enhance the total EUV irradiance flare variation by a factor of 2 when the late phase contribution is included.

Woods, Thomas N.↗

NASA NTRS2002

TIMED Solar EUV Experiment: Phase E

The timed Solar EUV Experiment (SEE) Phase E Annual Report for 2002 is presented. The contents include: 1) SEE Science Overview; 2) SEE Instrument Overview and Status; 3) Summary of SEE Data Products; 4) Summary of SEE Results; 5) Summary of SEE Related Talks and Papers; and 6) Future Plans for SEE Team. This paper is in viewgraph form.

Woods, Tom↗