Crystal structure of the complex of DNA with the C-terminal domain of TYE7 from Saccharomyces cerevisiae

TYE7, a bHLH (basic helix–loop–helix) transcription factor from Saccharomyces cerevisiae, is involved in the regulation of many genes, including glycolytic genes. Meanwhile, accumulating evidence indicates that TYE7 also functions as a cyclin and is linked to sulfur metabolism. Here, the structure of TYE7 (residues 165–291) complexed with its specific DNA was determined by X-ray crystallography. Structural analysis and comparison revealed that His185 and Glu189 are conserved in base recognition. However, Arg193 is also involved in base recognition in the structures that were compared. In the structure in this study, Arg193 in chain A has two conformations and makes a salt bridge with the phosphate backbone structure. In addition, a series of corresponding electrophoretic mobility shift assays were performed to better understand the DNA-binding mechanism of the bHLH domain of TYE7.

Download Full-text

Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0805642105 ◽

2008 ◽

Vol 105 (46) ◽

pp. 17682-17687 ◽

Cited By ~ 16

Author(s):

Jessica Bockhorn ◽

Bharvi Balar ◽

Dongming He ◽

Eden Seitomer ◽

Paul R. Copeland ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Null Allele ◽

Cost Effective ◽

Anomalous Dispersion ◽

Gene Encoding ◽

X Ray ◽

X Ray Crystallography ◽

Genome Wide ◽

Cost Effective Method ◽

Strain Collection

Selenomethionine (SeMet) is a potentially toxic amino acid, and yet it is a valuable tool in the preparation of labeled proteins for multiwavelength anomalous dispersion or single-wavelength anomalous dispersion phasing in X-ray crystallography. The mechanism by which high levels of SeMet exhibits its toxic effects in eukaryotic cells is not fully understood. Attempts to use Saccharomyces cerevisiae for the preparation of fully substituted SeMet proteins for X-ray crystallography have been limited. A screen of the viable S. cerevisiae haploid null allele strain collection for resistance to SeMet was performed. Deletion of the CYS3 gene encoding cystathionine gamma-lyase resulted in the highest resistance to SeMet. In addition, deletion of SSN2 resulted in both increased resistance to SeMet as well as reduced levels of Cys3p. A methionine auxotrophic strain lacking CYS3 was able to grow in media with SeMet as the only source of Met, achieving essentially 100% occupancy in total proteins. The CYS3 deletion strain provides advantages for an easy and cost-effective method to prepare SeMet-substituted protein in yeast and perhaps other eukaryotic systems.

Download Full-text

Role of folding kinetics of secondary structures in telomeric G-overhangs in the regulation of telomere maintenance in Saccharomyces cerevisiae

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012914 ◽

2020 ◽

Vol 295 (27) ◽

pp. 8958-8971 ◽

Cited By ~ 3

Author(s):

Katarina Jurikova ◽

Martin Gajarsky ◽

Mona Hajikazemi ◽

Jozef Nosek ◽

Katarina Prochazkova ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Secondary Structures ◽

Telomere Maintenance ◽

Folding Kinetics ◽

Mobility Shift ◽

Dna Structures ◽

Electrophoretic Mobility Shift Assays ◽

Kinetics Of

The ends of eukaryotic chromosomes typically contain a 3′ ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing–based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.

Download Full-text

Mutation of High-Affinity Methionine Permease Contributes to Selenomethionyl Protein Production in Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.01026-10 ◽

2010 ◽

Vol 76 (19) ◽

pp. 6351-6359 ◽

Cited By ~ 7

Author(s):

Toshihiko Kitajima ◽

Yasunori Chiba ◽

Yoshifumi Jigami

Keyword(s):

Saccharomyces Cerevisiae ◽

Methylotrophic Yeast ◽

Culture Conditions ◽

Crystallographic Analysis ◽

Gene Encoding ◽

X Ray ◽

X Ray Crystallography ◽

High Affinity ◽

Standard Culture ◽

Resistant Mutants

ABSTRACT The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.

Download Full-text

Purification of transmembrane proteins from Saccharomyces cerevisiae for X-ray crystallography

Protein Expression and Purification ◽

10.1016/j.pep.2009.12.012 ◽

2010 ◽

Vol 71 (2) ◽

pp. 207-223 ◽

Cited By ~ 27

Author(s):

Kathleen M. Clark ◽

Nadia Fedoriw ◽

Katrina Robinson ◽

Sara M. Connelly ◽

Joan Randles ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Transmembrane Proteins ◽

X Ray ◽

X Ray Crystallography

Download Full-text

A new view on crystal harvesting

Journal of Applied Crystallography ◽

10.1107/s1600576714008899 ◽

2014 ◽

Vol 47 (3) ◽

pp. 1158-1161 ◽

Cited By ~ 1

Author(s):

Joseph R. Luft ◽

Thomas D. Grant ◽

Jennifer R. Wolfley ◽

Edward H. Snell

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

High Throughput Screening ◽

Scale Up ◽

Trna Synthetase ◽

Objective Lens ◽

X Ray ◽

X Ray Crystallography ◽

Automated Process ◽

Structural Solution

X-ray crystallography typically requires the mounting of crystals, which can make the sample difficult to manipulate when it is small and the microscope objective is close to the crystallization plate. By simply moving the objective to the bottom of a clear crystallization plate (inverting the normal view), crystals were able to be manipulated and harvested from wells having a 0.9 mm diameter and 5.0 mm depth. The mounting system enabled the structural solution of the 187 amino acid N-terminal domain ofSaccharomyces cerevisiaeglutaminyl-tRNA synthetase from crystals that appeared during high-throughput screening but proved recalcitrant to scale-up and optimization. While not a general mounting solution, the simple expedient of removing the objective lens from the area where manipulation and harvesting occur greatly facilitates the manual, or even automated, process.

Download Full-text

Surface salt bridges modulate the DNA site size of bacterial histone-like HU proteins

Biochemical Journal ◽

10.1042/bj20050274 ◽

2005 ◽

Vol 390 (1) ◽

pp. 49-55 ◽

Cited By ~ 17

Author(s):

Edwin Kamau ◽

Nick D. Tsihlis ◽

L. Alice Simmons ◽

Anne Grove

Keyword(s):

Molecular Basis ◽

Genomic Dna ◽

Salt Bridge ◽

Salt Bridges ◽

Mobility Shift ◽

Dna Compaction ◽

Hu Proteins ◽

Optimal Leverage ◽

Electrophoretic Mobility Shift Assays ◽

Dna Cyclization

Bacterial histone-like DNA-binding proteins are best known for their role in compacting the genomic DNA. Of these proteins, HU is ubiquitous and highly conserved across the eubacterial kingdom. Using the HBsu (Bacillus subtilis-encoded HU homologue) as a model, we explore here the molecular basis for the ability of some HU homologues to engage a longer approx. 35 bp DNA site as opposed to the much shorter sites reported for other homologues. Using electrophoretic mobility-shift assays, we show that the DNA site size for HBsu is approx. 10–13 bp and that a specific surface salt bridge limits the DNA site size for HBsu. Surface exposure of the highly conserved Lys3, achieved by substitution of its salt-bridging partner Asp26 with Ala, leads to enhanced DNA compaction by HBsu-D26A (where D26A stands for the mutant Asp26→Ala), consistent with the interaction of Lys3 with the ends of a 25 bp duplex. Both HBsu and HBsu-D26A bend DNA, as demonstrated by their equivalent ability to promote ligase-mediated DNA cyclization, indicating that residues involved in mediating DNA kinks are unaltered in the mutant protein. We suggest that Lys3 is important for DNA wrapping due to its position at a distance from the DNA kinks where it can exert optimal leverage on flanking DNA and that participation of Lys3 in a surface salt bridge competes for its interaction with DNA phosphates, thereby reducing the occluded site size.

Download Full-text

Investigating Saccharomyces cerevisiae alkene reductase OYE 3 by substrate profiling, X-ray crystallography and computational methods

Catalysis Science & Technology ◽

10.1039/c8cy00440d ◽

2018 ◽

Vol 8 (19) ◽

pp. 5003-5016 ◽

Cited By ~ 2

Author(s):

Robert W. Powell, III ◽

M. Pilar Buteler ◽

Sunidhi Lenka ◽

Michele Crotti ◽

Sara Santangelo ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Computational Methods ◽

X Ray ◽

X Ray Crystallography ◽

Sequence Identity

Saccharomyces cerevisiae OYE 3 and OYE 1 share 80% sequence identity, but sometimes differ in stereoselectivities.

Download Full-text

Learning torus PCA based classification for multiscale RNA backbone structure correction with application to SARS-CoV-2

10.1101/2021.08.06.455406 ◽

2021 ◽

Author(s):

Henrik Wiechers ◽

Benjamin Eltzner ◽

Kanti V. Mardia ◽

Stephan F. Huckemann

Keyword(s):

Correction Method ◽

Training Data ◽

Proof Of Concept ◽

Data Set ◽

X Ray ◽

X Ray Crystallography ◽

Rna Backbone ◽

Backbone Structure ◽

Mode Hunting ◽

Structure Enhancement

Reconstructions of structure of biomolecules, for instance via X-ray crystallography or cryo-EM frequently contain clashes of atomic centers. Correction methods are usually based on simulations approximating biophysical chemistry, making them computationally expensive and often not correcting all clashes. We propose a computationally fast data-driven statistical method yielding suites free from within-suite clashes: From such a clash free training data set, devising mode hunting after torus PCA on adaptive cutting average linkage tree clustering (MINTAGE), we learn RNA suite shapes. With classification based on multiscale structure enhancement (CLEAN), for a given clash suite we determine its neighborhood on a mesoscopic scale involving several suites. As corrected suite we propose the Fréchet mean on a torus of the largest classes in this neighborhood. We validate CLEAN MINTAGE on a benchmark data set, compare it to a state of the art correction method and apply it, as proof of concept, to two exemplary suites adjacent to helical pieces of the frameshift stimulation element of SARS-CoV-2 which are difficult to reconstruct. In contrast to a recent reconstruction proposing several different structure models, CLEAN MINTAGE unanimously proposes structure corrections within the same clash free class for all suites.

Download Full-text

A Chemical Probe for Dark Kinase STK17B Derives its Potency and High Selectivity Through a Unique P-loop Conformation

10.26434/chemrxiv.12616178.v1 ◽

2020 ◽

Author(s):

Alfredo Picado ◽

Apirat Chaikuad ◽

Carrow Wells ◽

Safal Shrestha ◽

William Zuercher ◽

...

Keyword(s):

Carboxylic Acid ◽

High Selectivity ◽

Salt Bridge ◽

Competitive Inhibitor ◽

Chemical Probe ◽

High Quality ◽

X Ray ◽

X Ray Crystallography ◽

Loop Conformation

We present the discovery of thieno[3,2-d]pyrimidine SGC-STK17B-1 (11s), a high-quality chemical probe for this understudied “dark” kinase. 11s is an ATP-competitive inhibitor that showed remarkable selectivity over other kinases including the closely related STK17A. X-ray crystallography of 11s and related thieno[3,2-d]pyrimidines bound to STK17B revealed a unique P-loop conformation characterized by a salt bridge between R41 and the carboxylic acid of the inhibitor.

Download Full-text

Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C4 photosynthesis is based on an ancient code found in the ancestral C3 state

10.1101/230136 ◽

2018 ◽

Author(s):

Ana Rita Borba ◽

Tânia S. Serra ◽

Alicja Górska ◽

Paulo Gouveia ◽

André M. Cordeiro ◽

...

Keyword(s):

Transcription Factors ◽

Regulatory Networks ◽

Molecular Mechanisms ◽

Mobility Shift ◽

Helix Loop Helix ◽

Specific Accumulation ◽

Cell Type Specific ◽

Bhlh Transcription Factors ◽

Electrophoretic Mobility Shift Assays ◽

Element Pair

AbstractC4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialised form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world’s most productive crops. The C4 cycle is accomplished through cell-type specific accumulation of enzymes but cis-elements and transcription factors controlling C4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we aimed to better understand molecular mechanisms associated with the evolution of C4 photosynthesis. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C4NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C3 and C4 species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C3 state. We conclude that C4 photosynthesis has co-opted an ancient C3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C4 photosynthesis.

Download Full-text