Biology subjects

Landick, R.

Publications and source records attributed to Landick, R..

5 recordsLinked to original sources

bioRxiv2018

RNA polymerase clamp movement aids dissociation from DNA but is not required for RNA release at intrinsic terminators

In bacteria, disassembly of elongating transcription complexes (ECs) can occur at intrinsic terminators in a 2-3 nucleotide window after transcription of multiple kilobase pairs of DNA. Intrinsic terminators trigger pausing on weak RNA-DNA hybrids followed by formation of a strong, GC-rich stem-loop in the RNA exit channel of RNA polymerase (RNAP), inactivating nucleotide addition and inducing dissociation of RNA and RNAP from DNA. Although the movements of RNA and DNA during intrinsic termination have been studied extensively leading to multiple models, the effects of RNAP conformational changes remain less well-defined. RNAP contains a clamp domain that closes around the nucleic-acid scaffold during transcription initiation and can be displaced by either swiveling or opening motions. Clamp opening is proposed to promote termination by releasing RNAP-nucleic acid contacts. We developed a cysteine-crosslinking assay to constrain clamp movements and study effects on intrinsic termination. We found that biasing the clamp into different conformations perturbed termination efficiency, but that perturbations were due primarily to changes in elongation rate, not the competing rate at which ECs commit to termination. After commitment, however, inhibiting clamp movements slowed release of DNA but not of RNA from the EC. We also found that restricting trigger-loop movements with the RNAP inhibitor microcin J25 prior to commitment inhibits termination, in agreement with a recently proposed multistate-multipath model of intrinsic termination. Together our results support views that termination commitment and DNA release are separate steps and that RNAP may remain associated with DNA after termination.\n\nHighlightsO_LIDisulfide bond crosslinks probe the role of the RNAP clamp domain in termination\nC_LIO_LIRNA but not DNA can release at terminators when the RNAP clamp is closed\nC_LIO_LIRestricting RNAP clamp movement affects elongation rate more than termination rate\nC_LIO_LIInhibiting TL conformational flexibility impairs both RNA and DNA release\nC_LI

molecular biology↗

bioRxiv2018

The elemental mechanism of transcriptional pausing

Transcriptional pausing underpins regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) can easily enter pretranslocated and one-base-pair backtracked states despite principally occupying the half-translocated state observed in cryo-EM structures of paused RNAPs. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators.

biochemistry↗

bioRxiv2018

Structural basis for transcript elongation control by NusG/RfaH universal regulators

NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. In bacteria, NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and also coordinating {rho}-dependent termination and translation. RfaH is a specialized NusG paralog that engages the EC at ops sites and subsequently excludes NusG and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryo-EM to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base pairing of the EC upstream duplex DNA to suppress backtracking. RfaH loads onto the EC by specific recognition of an ops hairpin in the single-stranded nontemplate DNA. Binding of both NusG and RfaH is incompatible with the swiveled RNAP conformation necessary for hairpin-stabilized pausing, but only RfaH fully counteracts swiveling to suppress pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.

biophysics↗

bioRxiv2018

Fidaxomicin jams M. tuberculosis RNA polymerase motions needed for initiation via RbpA contacts

Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against Mycobacterium tuberculosis RNAP in vitro, but clinical use of Fdx is limited to treating Clostridium difficile intestinal infections due to poor absorption. To enable structure-guided optimization of Fdx to treat tuberculosis, we report the 3.4 [A] cryo-electron microscopy structure of a complete M. tuberculosis RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin and explains its strong effect on M. tuberculosis. We present additional structures that define conformational states of M. tuberculosis RNAP between the free apo-holenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on Fdx.

biophysics↗

bioRxiv2017

Chemical genomic guided engineering of gamma-valerolactone tolerant yeast

BackgroundGamma valerolactone (GVL) is a promising technology for degradation of biomass for biofuel production; however, GVL has adverse toxicity effects on fermentative microbes. Using a combination of chemical genomics and chemical proteomics we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast.\n\nResultsChemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate phenolic acids to vinyl derivatives, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were more abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain.\n\nConclusionsWe found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol content in cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain. This strain represents a xylose fermenting yeast specifically tailored to GVL produced hydrolysates

bioengineering↗