Ailong Ke - Academia.edu (original) (raw)

Papers by Ailong Ke

Molecular cell, Jan 19, 2015

Gene regulation in cis by riboswitches is prevalent in bacteria. The yybP-ykoY riboswitch family ... more Gene regulation in cis by riboswitches is prevalent in bacteria. The yybP-ykoY riboswitch family is quite widespread, yet its ligand and function remained unknown. Here, we characterize the Lactococcus lactis yybP-ykoY orphan riboswitch as a Mn(2+)-dependent transcription-ON riboswitch, with a ∼30-40 μM affinity for Mn(2+). We further determined its crystal structure at 2.7 Å to elucidate the metal sensing mechanism. The riboswitch resembles a hairpin, with two coaxially stacked helices tethered by a four-way junction and a tertiary docking interface. The Mn(2+)-sensing region, strategically located at the highly conserved docking interface, has two metal binding sites. Whereas one site tolerates the binding of either Mg(2+) or Mn(2+), the other site strongly prefers Mn(2+) due to a direct contact from the N7 of an invariable adenosine. Mutagenesis and a Mn(2+)-free E. coli yybP-ykoY structure further reveal that Mn(2+) binding is coupled with stabilization of the Mn(2+)-sensing reg...

Protein science : a publication of the Protein Society, 2003

The Yeast MATa1 and MATalpha2 are homeodomain proteins that bind DNA cooperatively to repress tra... more The Yeast MATa1 and MATalpha2 are homeodomain proteins that bind DNA cooperatively to repress transcription of cell type specific genes. The DNA affinity and specificity of MATa1 in the absence of MATalpha2, however, is very low. MATa1 is converted to a higher affinity DNA-binding protein by its interaction with the C-terminal tail of MATalpha2. To understand why MATa1 binds DNA weakly by itself, and how the MATalpha2 tail affects the affinity of MATa1 for DNA, we determined the crystal structure of a maltose-binding protein (MBP)-a1 chimera whose DNA binding behavior is similar to MATa1. The overall MATa1 conformation in the MBP-a1 structure, which was determined in the absence of alpha2 and DNA, is similar to that in the a1/alpha2/DNA structure. The sole difference is in the C-terminal portion of the DNA recognition helix of MATa1, which is flexible in the present structure. However, these residues are not in a location likely to be affected by binding of the MATalpha2 tail. The r...

Structure (London, England : 1993), 2002

Triply mutated MATalpha2 protein, alpha2-3A, in which all three major groove-contacting residues ... more Triply mutated MATalpha2 protein, alpha2-3A, in which all three major groove-contacting residues are mutated to alanine, is defective in binding DNA alone or in complex with Mcm1 yet binds with MATa1 with near wild-type affinity and specificity. To gain insight into this unexpected behavior, we determined the crystal structure of the a1/alpha2-3A/DNA complex. The structure shows that the triple mutation causes a collapse of the alpha2-3A/DNA interface that results in a reorganized set of alpha2-3A/DNA contacts, thereby enabling the mutant protein to recognize the wild-type DNA sequence. Isothermal titration calorimetry measurements reveal that a much more favorable entropic component stabilizes the a1/alpha2-3A/DNA complex than the alpha2-3A/DNA complex. The combined structural and thermodynamic studies provide an explanation of how partner proteins influence the sequence specificity of a DNA binding protein.

homeodomain bound to DNA, the a1/␣2 heterodimer bound to DNA, and the ␣2/MCM1 heterotetramer boun... more homeodomain bound to DNA, the a1/␣2 heterodimer bound to DNA, and the ␣2/MCM1 heterotetramer bound to DNA [9-11]. The DNA binding domain of ␣2 adopts a typical homeodomain fold [11][12][13], with a compact three-helix globular domain that contacts major groove bases and the DNA backbone, mainly through the third helix, and an extended N-terminal arm that inserts into School of Medicine 725 North Wolfe Street the DNA minor groove. The contacts between ␣2 and DNA are nearly identical in all three crystal structures. The ␣2 protein contacts four base pairs in the DNA major groove using three side chains in helix 3: Ser50, Asn51, Department of Biochemistry Rutgers University and Arg54 ). Ser50 forms two watermediated hydrogen bonds with A4 and T5, Arg54 con-Piscataway, New Jersey 08854 tacts G6 with two direct hydrogen bonds, and Asn51 forms bidentate hydrogen bonds with A38. Asn51, which is an invariant homeodomain residue, forms identical Summary contacts in other homeodomain-DNA structures [11][12][13]. Additional side chains in helix 3 of ␣2 make DNA Triply mutated MAT␣2 protein, ␣2-3A, in which all phosphate backbone contacts, which help to position three major groove-contacting residues are mutated the recognition helix of ␣2 in the DNA major groove. In to alanine, is defective in binding DNA alone or in com-

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2014

The recent discovery of short cis-acting RNA elements termed riboswitches has caused a paradigm s... more The recent discovery of short cis-acting RNA elements termed riboswitches has caused a paradigm shift in our understanding of genetic regulatory mechanisms. The three distinct superfamilies of S-adenosyl-l-methionine (SAM) riboswitches are the most commonly found riboswitch classes in nature. These RNAs represent three independent evolutionary solutions to achieve specific SAM recognition. This review summarizes research on 1) modes of gene regulatory mechanisms, 2) common themes and differences in ligand recognition, and 3) ligand-induced conformational dynamics among SAM riboswitch families. The body of work on the SAM riboswitch families constitutes a useful primer to the topic of gene regulatory RNAs as a whole. This article is part of a Special Issue entitled: Riboswitches.

Journal of Molecular Biology, 2015

Nature Structural & Molecular Biology, 2008

Three distinct classes of S-adenosyl-L-methionine (SAM)-responsive riboswitches have been identif... more Three distinct classes of S-adenosyl-L-methionine (SAM)-responsive riboswitches have been identified that regulate bacterial gene expression at the levels of transcription attenuation or translation inhibition. The SMK box (SAM-III) translational riboswitch has been identified in the SAM synthetase gene in members of the Lactobacillales. Here we report the 2.2-Å crystal structure of the Enterococcus faecalis SMK box riboswitch. The Y-shaped riboswitch

Nature Structural & Molecular Biology, 2014

7 7 1 a r t i c l e s Clustered regularly interspaced palindromic repeats (CRISPR) drives adaptat... more 7 7 1 a r t i c l e s Clustered regularly interspaced palindromic repeats (CRISPR) drives adaptation to invasive nucleic acids such as phages, conjugative plasmids and transposable elements, using an RNA-mediated interference mechanism that has fundamental similarities to innate and adaptive immune responses 1-3 . This RNA-based adaptive immunity system involves a noncoding CRISPR array and a nearby CRISPRassociated (cas) operon 4-6 . CRISPR-Cas systems mediate three key molecular events: (i) adaptation through the insertion of short segments of 'spacer DNA' derived from foreign genetic elements into the CRISPR array; (ii) transcription of the CRISPR array and endoribonucleolytic processing of transcripts into CRISPR RNA (crRNA); and (iii) crRNA-guided degradation of the foreign DNA 7 containing spacer-complementary sequences 1-3 . (RNAs are targeted in type III-B CRISPR systems, as exemplified in Sulfolobus solfataricus and Pyrococcus furiosus 8 .)

We have expanded the application of antibody phage display to a new type of antigen: ribonucleopr... more We have expanded the application of antibody phage display to a new type of antigen: ribonucleoprotein (RNP) complexes. We describe a simple and efficient method for screening antibodies specific for large intact RNPs and individual components. We also describe a fast and easy method to overcome the abundance of amber stop codons in the positive phage clones. The resulting antibodies have been used in ELISA and Western blot analysis.

Structure, 2007

The hepatitis delta virus (HDV) ribozyme catalyzes viral RNA self-cleavage through general acid-b... more The hepatitis delta virus (HDV) ribozyme catalyzes viral RNA self-cleavage through general acid-base chemistry in which an active-site cytidine and at least one metal ion are involved. Monovalent metal ions support slow catalysis and were proposed to substitute for structural, but not catalytic, divalent metal ions in the RNA. To investigate the role of monovalent cations in ribozyme structure and function, we determined the crystal structure of the precursor HDV ribozyme in the presence of thallium ions (Tl + ). Two Tl + ions can occupy a previously observed divalent metal ion hexahydrate-binding site located near the scissile phosphate, but are easily competed away by cobalt hexammine, a magnesium hexahydrate mimic and potent reaction inhibitor. Intriguingly, a third Tl + ion forms direct inner-sphere contacts with the ribose 2 0 -OH nucleophile and the pro-S p scissile phosphate oxygen. We discuss possible structural and catalytic implications of monovalent cation binding for the HDV ribozyme mechanism.

Methods, 2004

RNA plays a direct role in a variety of cellular activities, and in many cases its biological fun... more RNA plays a direct role in a variety of cellular activities, and in many cases its biological function is conferred by the RNA three-dimensional structure. X-ray crystallography is the method of choice for determining high resolution structures of large RNA molecules, and can also be used to compare related RNAs and identify conformational changes that may accompany biochemical activity. However, crystallization remains the rate-limiting step in RNA structure determination due to the difficulty in obtaining well-ordered crystals for X-ray diffraction analysis. Several approaches to sample preparation, crystallization, and crystal handling are presented that have been used successfully in the structure determination of RNA and RNA-protein complexes in our laboratory, and should be generally applicable to RNAs in other systems.

RNA Biology, 2013

The term riboswitch usually refers to small molecule sensing regulatory modules in the 5&... more The term riboswitch usually refers to small molecule sensing regulatory modules in the 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; untranslated regions of a mRNA. They are typically comprised of separate ligand binding and regulatory domains. The T box riboswitch is unique from other identified riboswitches because its effector is an essential macromolecule, tRNA. It senses the aminoacylation state of tRNA to regulate genes involved in a variety of functions relating to amino acid metabolism and tRNA aminoacylation. T box riboswitches performs an intuitively simple process using a complex structured RNA element and, until recently, the underlying mechanisms were poorly understood. Only two sequence-specific contacts had been previously identified: (1) between the specifier sequence (codon) and the tRNA anticodon and (2) between an anti-terminator stem loop and the tRNA acceptor arm CCA tail. tRNA aminoacylation blocks the latter interaction and therefore serves as the switch between termination and anti-termination. Outside of these two contacts, the structure and functions of T box riboswitches have come to light in some recent studies. We recently described the X-ray crystal structure of the highly conserved T box riboswitch distal Stem I region and demonstrated that this region interacts with the tRNA elbow to anchor it to the riboswitch. Independently, Lehmann et al. used sequence homology search to arrive at a similar model for Stem I-tRNA interactions. The model was further supported by two recent structures of the Stem I-tRNA complex, determined independently by our group and by Zhang and Ferré-D&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;Amaré. This article highlights some of these contributions to synthesize an updated model for tRNA recognition by the T box riboswitch.

Proceedings of the National Academy of Sciences, 2011

the paper.

PLoS ONE, 2010

Background: The exosome complex is an essential RNA 39-end processing and degradation machinery. ... more Background: The exosome complex is an essential RNA 39-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry.

Nature Structural & Molecular Biology, 2005

The signal recognition particle (SRP) targets nascent proteins to cellular membranes for insertio... more The signal recognition particle (SRP) targets nascent proteins to cellular membranes for insertion or secretion by recognizing polypeptides containing an N-terminal signal sequence as they emerge from the ribosome. GTP-dependent binding of SRP to its receptor protein leads to controlled release of the nascent chain into a membrane-spanning translocon pore. Here we show that the association of the SRP with its receptor triggers a marked conformational change in the complex, localizing the SRP RNA and the adjacent signal peptide-binding site at the SRP-receptor heterodimer interface. The orientation of the RNA suggests how peptide binding and GTP hydrolysis can be coupled through direct structural contact during cycles of SRP-directed protein translocation.

Nature Structural & Molecular Biology, 2013

The eukaryotic exosome is a multi-subunit complex typically composed of a catalytically inactive ... more The eukaryotic exosome is a multi-subunit complex typically composed of a catalytically inactive core and the Rrp44 protein, which contains 3' to 5' exo-and endo-ribonuclease activities. RNA substrates have been shown to be recruited through the core to reach Rrp44's exoribonuclease (EXO) site. Using single particle electron microscopy and biochemical analysis, we provide visual evidence that two distinct substrate recruitment pathways exist. In the through-core route, channeling of the single stranded substrates from the core to Rrp44 induces a characteristic conformational change in Rrp44. In the alternative direct-access route, this conformational change does not take place and the RNA substrate is visualized to avoid the core and enter Rrp44's EXO site directly. Our results provide mechanistic explanations for several RNA processing scenarios by the eukaryotic exosome and indicate substrate specific modes of degradation by this complex.

Molecular Microbiology, 2011

A widespread feature in the genomes of most bacteria and archaea is an array of clustered, regula... more A widespread feature in the genomes of most bacteria and archaea is an array of clustered, regularly interspaced short palindromic repeats (CRISPRs) that, together with a group of CRISPR-associated (Cas) proteins, mediate immunity against invasive nucleic acids such as plasmids and viruses. Here, the CRISPR-Cas system was activated in cells expressing a plasmid-encoded protein that was targeted to the twin-arginine translocation (Tat) pathway. Expression of this Tat substrate resulted in upregulation of the Cas enzymes and subsequent silencing of the encoding plasmid in a manner that required the BaeSR two-component regulatory system, which is known to respond to extracytoplasmic stress. Furthermore, we confirm that the CasCDE enzymes form a stable ternary complex and appear to function as the catalytic core of the Cas system to process CRISPR RNA into its mature form. Taken together, our results indicate that the CRISPR-Cas system targets DNA directly as part of a defence mechanism in bacteria that is overlapping with but not limited to phage infection.