| 1. |
- Lüking, Malin, et al.
(författare)
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Molecular Origins of Kinetics and Selectivity in the lac operon
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- We address the question of specificity in protein-DNA interactions using the E. colitranscription factor LacI as an example. Switching between two conformations, one fornon-specific, transient and another for specific, long-lived interaction with DNA, has beensuggested to help DNA-binding proteins solve the conflict between fast search andstable, specific binding. We tested this idea by changing the ability of LacI to switchconformations. We used molecular simulation to select LacI variants with altered flexibilityin the region that is involved in LacI’s conformational change, the hinge helix. We thenused fluorescent microscopy to study the wild-type LacI and LacI variants when binding tonon-operator and operator-DNA in vivo. In fact, LacI with a more flexible hinge helix is aweaker binder that exhibits less off-target site binding. A more stable helix, in contrast,enhances the formation of long-lived protein-DNA complexes also with non-operatorDNA. We examined the effects of two allosteric factors of LacI, the inducer IPTG, whichreduces DNA affinity, and the ligand ONPG, which enhances DNA binding according toour finding. We found that wild-type LacI is optimised for high stability of the specificcomplex and sensitivity to induction.
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| 2. |
- Marklund, Emil, et al.
(författare)
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Sequence specificity in DNA binding is mainly governed by association
- 2022
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 375:6579, s. 442-445
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Tidskriftsartikel (refereegranskat)abstract
- Sequence-specific binding of proteins to DNA is essential for accessing genetic information. We derive a model that predicts an anticorrelation between the macroscopic association and dissociation rates of DNA binding proteins. We tested the model for thousands of different lac operator sequences with a protein binding microarray and by observing kinetics for individual lac repressor molecules in single-molecule experiments. We found that sequence specificity is mainly governed by the efficiency with which the protein recognizes different targets. The variation in probability of recognizing different targets is at least 1.7 times as large as the variation in microscopic dissociation rates. Modulating the rate of binding instead of the rate of dissociation effectively reduces the risk of the protein being retained on nontarget sequences while searching.
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| 3. |
- Vogel, Carolin, et al.
(författare)
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Rationally designed Spot 42 RNAs with an inhibition/toxicity profile advantageous for engineering E. coli
- 2020
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Ingår i: ENGINEERING REPORTS. - : Wiley. - 2577-8196. ; 2:3
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial regulatory small RNAs (sRNAs) have shown promise for gene knock-down studies and metabolic engineering. However, some mRNAs might be difficult to target due to poor binding by the Hfq chaperone, individual synthetic sRNAs can have off-target effects, potential sRNA toxicities have not been studied globally, and a consensus on optimal sRNA design has yet to emerge. Here, Spot 42 sRNA is validated as an excellent scaffold by showing that its over-expression minimally affects the growth rate of Escherichia coli, and that inhibition is reliably achieved for all eight tested protein targets by designing antisense to target the first few codons. Two related sRNAs that could not be cloned, possibly due to lethality of the encoded sRNAs, became clonable when an eight-nucleotide sequence was inserted directly upstream of the antisense region. Global fitness costs for E. coli of the designer sRNAs were measured and found to be variable but tolerable. Importantly for utility, there was no correlation between target inhibition and cellular toxicity. As a proof of concept for applications, suppression of the UAG stop codon was improved by knock down of translation release factor 1 (RF1).
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