Name: site-directed mutagenesis for in vitro and in vivo experiments exemplified with rna interactions in escherichia coli
Uploaded: 2019-02-05T15:00:00
Description: Watch this Scientific Journal Video about Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli at JoVE.com

The authors would like to thank University of Southern Denmark open access policy grants.

1. Vector selection
<ol>
	<li>Choose a vector to perform downstream experiments with. Any vector is applicable for this 2- and 3-step PCR method.</li>
	<li>Based on choice of vector, choose appropriate restriction enzymes for cloning.</li>
</ol>
2. Primer design for site directed mutagenesis
<ol>
	<li>Decide between either the 2-step or 3-step PCR strategy (2-step is only for mutations &#60;200 bp from either end of the DNA of interest). For the 2-step PCR, go to step 2.2 and for the 3-step PC...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cassette+mutagenesis:+an+efficient+method+for+generation+of+multiple+mutations+at+defined+sites.">Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites.</a> Gene. 34 (2-3), 315-323 (1985).</li><li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li></ol>

Site-directed mutagenesis has a broad array of different applications, and here, representative results from an in vivo and an in vitro experiment were included as examples of how to make biological conclusions using the technique. Site-directed mutagenesis has for long been the golden standard for RNA interaction studies. The strength of the technique lies in the combination of introducing relevant mutations with downstream assays and experiments (e.g., western blot or EMSA) to draw conclusions about specific DNA sites ...

DNA is often referred to as the blueprint of a living cell since all structures of the cell are encoded in the sequence of its DNA. Accurate replication and DNA repair mechanisms ensure that only very low rates of mutations occur, which is essential for sustaining correct functions of coded genes. Changes of the DNA sequence can affect successive functions at different levels starting with DNA (recognition by transcription factors and restriction enzymes), then RNA (base-pair complementarity and secondary structure alterations) and/or protein (amino acid substitutions, deletions, additions or frame-shifts). While many mutations do not affect gene function significantly, some mutations in the DNA can have huge implications. Thus, site-directed mutagenesis is a valuable tool for studying the importance of specific DNA sites at all levels.
This protocol describes a targeted mutagenesis approach used to introduce specific mutations. The protocol relies on two different PCR strategies: a 2-step or a 3-step PCR. The 2-step PCR is applicable if the desired mutation is close to either the 5&#39; end or the 3&#39; end of the DNA of interest (&#60;200 base pairs (bp) from the end) and the 3-step PCR is applicable in all cases.
In the 2-step PCR approach, 3 primers are designed, in which one set of primers is designed to amplify the DNA of interest (primers 1 and 3, forward and reverse, respectively), and a single primer is designed to incorporate the mutation. The mutation introducing primer (primer 2) should have a reverse orientation if the mutation is close to the 5&#39; end and a forward orientation if the mutation is close to the 3&#39; end. In the first PCR step, primer 1+2 or 2+3 amplifies a small fragment close to the 5&#39; end or 3&#39; end, respectively. The resulting PCR product is then used as a primer in step two with primer 1 or 3, thus resulting in a PCR product with a mutation in the DNA of interest (Figure 1A).
In the 3-step PCR, 4 primers are designed, in which one set of primers is designed to amplify the DNA of interest (primers 1 and 4, forward and reverse, respectively) and one set of primers is designed to incorporate specific mutations with overlapping complementarity (primers 2 and 3, reverse and forward, respectively). In step one and two, primers 1+2 and 3+4 amplify the 5&#39; and 3&#39; end. In step three, the resulting PCR products from step one and two are used as templates and amplified with primers 1+4. Thus, the resulting PCR product is the DNA of interest with the desired mutation (Figure 1B).
While the mutated DNA can be used for any downstream application, this protocol describes how to re-combine the DNA into a cloning vector. The use of cloning vectors has several advantages such as ease of cloning and specific experimental applications depending on features of the vector. This feature is often used for RNA interaction studies. Another technique for RNA interaction studies is structural probing of the RNA in complex with another RNA1,2 or protein3,4. However, structural probing is only performed in vitro whereas site-directed mutagenesis and subsequent cloning allow for interaction studies in vivo.
Site-directed mutagenesis has been extensively used for RNA interaction studies as presented here. However, the key method regarding 2- or 3-step PCR is applicable to any piece of DNA, and thus not only limited to RNA-interaction studies.
To exemplify the technique and its possible uses, characterization of regions important for post-transcriptional regulation of the mRNA, csgD, of Escherichia coli (E. coli) is used. In E. coli, csgD is targeted by the small non-coding RNA, McaS, in cooperation with a protein, Hfq, to repress protein-expression of CsgD2,4,5. The technique is used to introduce mutations to the base-pairing region between csgD and McaS, and to the Hfq binding site of csgD. The obtained DNA is then cloned into a vector suitable for subsequent experiments. Downstream applications of the technique include both in vivo and in vitro experiments. For illustration, example 1 is characterized in vivo using a western blot assay and example 2 is characterized in vitro using an electrophoretic mobility shift assay (EMSA). In both cases, it is illustrated how site-directed mutagenesis can be used in combination with other techniques to make biological conclusions about a gene of interest.

<table>
	<tbody>
		<tr>
			<td>Anti-GroEL antibody produced in rabbit</td>
			<td>Merck</td>
			<td>G6532</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Azure c200</td>
			<td>Azure</td>
			<td>NA</td>
			<td>Gel imaging workstation</td>
		</tr>
		<tr>
			<td>Custom DNA oligo</td>
			<td>Merck</td>
			<td>VC00021</td>
			<td></td>
		</tr>
		<tr>
			<td>DeNovix DS-11</td>
			<td>DeNovix</td>
			<td>NA</td>
			<td>Spectrophotometer for nucleic acid measurements</td>
		</tr>
		<tr>
			<td>DNA Gel Loading Dye (6X)</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution 1 %</td>
			<td>Carl Roth</td>
			<td>2218.1</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneJET Gel Extraction Kit</td>
			<td>Thermo Scientific</td>
			<td>K0691</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler DNA Ladder Mix</td>
			<td>Fermentas</td>
			<td>SM0333</td>
			<td></td>
		</tr>
		<tr>
			<td>Gerard GeBAflex-tube Midi</td>
			<td>Gerard Biotech</td>
			<td>TO12</td>
			<td>Dialysis tubes for electro elution</td>
		</tr>
		<tr>
			<td>MEGAscript T7 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Sub Cell GT Cell</td>
			<td>Bio-Rad</td>
			<td>1704406</td>
			<td>Horizontal electrophoresis system</td>
		</tr>
		<tr>
			<td>Monoclonal ANTI-FLAG M2 antibody produced in mouse</td>
			<td>Merck</td>
			<td>F3165</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Mouse Immunoglobulins</td>
			<td>Dako Cytomation</td>
			<td>P0447</td>
			<td>HRP conjucated secondary antibody</td>
		</tr>
		<tr>
			<td>NucleoSpin miRNA</td>
			<td>Macherey Nagel</td>
			<td>740971</td>
			<td>RNA purification</td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td>Thermo Scientific</td>
			<td>NP0323BOX</td>
			<td>Bis-Tris gels for protein separation</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity PCR Master Mix with HF Buffer</td>
			<td>New England Biolabs</td>
			<td>M0531S</td>
			<td>DNA polymerase</td>
		</tr>
		<tr>
			<td>PowerPac HC High-Current Power Supply</td>
			<td>Bio-Rad</td>
			<td>1645052</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Immunoglobulins</td>
			<td>Dako Cytomation</td>
			<td>P0448</td>
			<td>HRP conjucated secondary antibody</td>
		</tr>
		<tr>
			<td>SeaKem LE Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>SigmaPlot</td>
			<td>Systat Software Inc</td>
			<td>NA</td>
			<td>Graph and data analysis software tool</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td>PCR machine</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202</td>
			<td>Ligase</td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE Buffer (Tris-acetate-EDTA) (50X)</td>
			<td>Thermo Scientific</td>
			<td>B49</td>
			<td></td>
		</tr>
	</tbody>
</table>

site-directed mutagenesis for in vitro and in vivo experiments exemplified with rna interactions in escherichia coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

To investigate RNA interactions regarding post-transcriptional regulation of csgD, a double vector setup was chosen: one to express the csgD mRNA and another to express the small non-coding RNA, McaS. csgD was cloned into pBAD33, which is an arabinose inducible medium-copy plasmid with chloramphenicol resistance and McaS was cloned into mini R1 pNDM220, which is an isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) inducible low copy plasmid with ampicillin resis...

Watch this Scientific Journal Video about Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli at JoVE.com

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Site-directed mutagenesis is useful to study molecular interactions, such as RNA-RNA, and RNA protein interactions. This protocol describes how to perform site-directed mutagenesis with a two or 3-step PCR approach. The main advantages of the method are the diversity of different mutations that can be incorporated, as well as a relative ease and cost for doing so.To begin, use the wild-type DNA template and primer pairs two, one, and two, three, specific for 2-step, or primer pairs three, one, and three, four, specific for 3-step PCR to obtain PCR product one. To validate PCR products by agarose gel electrophoresis, make a 2%agarose gel by dissolving two grams of agarose in 100 milliliters of 1X TAE buffer, using a microwave oven. Add ethidium bromide at a final concentration of 0.5 microgram per milliliter.Then cast the agarose gel. When solidified, place it in an electrophoresis unit. Load a DNA ladder of known size, and the PCR samples mixed with DNA loading dye into wells.Run samples at 75 volts for 45 minutes. And visualize bands on a UV transilluminator or with a gel imaging system. Next, purify the PCR product with a gel extraction kit according to the manufacturers protocol and measure the concentration of the purified DNA with a spectrophotometer.Then store the purified DNA at minus 20 celsius in TE buffer. To begin site-directed mutagenesis with a 2-step PCR, first use the wild-type DNA template, then the primer pairs two, one and two, two, for five primer mutations, or primer pairs two, two, and two, three for three priment mutations to obtain PCR product two. Validate the PCR product by gel electrophoresis, purify the PCR product, and measure its concentration as described before.Then use the wild-type DNA template and the PCR product two as the primer, together with primer two, three for five priment mutation, or primer two, one for three priment mutation, to obtain PCR product three. Validate the PCR product by gel electrophoresis, purify the PCR product, and measure its concentration as described before. Then store the purified DNA at minus 20 degrees celsius in TE buffer.To begin site-direct mutagenesis with a 3-step PCR, first use the wild-type DNA template and primer pairs three, one, and three, two to obtain PCR product two. Use the wild-type DNA template and primer pairs three, three, and three, four, to obtain PCR product three. Use the wild-type DNA template and primer pairs three, three and three, four, to obtain PCR product three.Validate the PCR product by gel electrophoresis, purify the PCR product, and measure its concentration as described before. Finally use two to five nanograms of PCR products two and three as the template along with primer pairs three, one and three, four, to obtain PCR product four. Validate the PCR product by gel electrophoresis, purify the PCR product, and measure its concentration as described before.Store the purified DNA at minus 20 degrees celsius in TE buffer. In this study, 2-step and 3-step site-directed mutagenesis were used to investigate RNA interactions regarding post transcriptional regulation of CsgD. The wild-type CsgD was PCR amplified to yield PCR product IA, and the wild-type mcaS was PCR amplified to yield PCR product IB.Using a 3-step PCR strategy, PCR products IIA and IIIA were synthesized in the first two steps, and IVA in the third step to introduce mutations in CsgD.Similarly, complimentary site-directed mutations were introduced in mcaS to yield PCR products IIB, IIIB, in the first two steps, and IVB in the third step. Western blot analysis showed that while expression of wild-type mcaS prevents translation of wild-type CsgD, mutations in either mcaS or CsgD alleviates the observed repression. However, mutations in both CsgD and mcaS prevent translation of CsgD.Using a 2-step PCR strategy, PCR products IIC, IID, IIE, IIF, and IIG, were synthesized in the first step, and PCR products IIIC, IIID, IIIE, IIIF, and IIIG, in the second step to introduce mutations to the entire CsgD DNA. EMSA results of the HFQ binding to in vitro transcribed CsgD wild-type and mutant RNAs, synthesized using PCR products IIIC, IIID, IIIE, IIIF, and IIIG, showed the increased KD values of the mutant alleles. This proved that HFQ could bind less efficiently to the mutants.Furthermore HFQ has several binding sites on CsgD, which is shown by the three shifts observed for the wild-type RNA. However, only two binding sites are observed for different mutant RNAs. Thus the site-directed mutational approach identifies primary and or secondary structures of the CsgD mRNA that are important for complete binding of HFQ.The method for site-directed mutagenesis described here relies on PCR. Good PCR practices are therefor crucial for the method and might require optimization to be successful. Site-directed mutagenesis is only powerful with all three methods such EMSA and Western Blotting.Other methods, for instance, include various structural assays and some activity assays, and post translation modification studies.

site-directed-mutagenesis-for-vitro-vivo-experiments-exemplified-with

Research

JoVE Journal

Biochemistry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.