Chemical Modification RNA Footprinting

Details of chemical modification footprinting as performed in our laboratory, a modification of procedures described by Moazed et al. (1986) and Christiansen et al (1990), are given below. (Note All solutions are made with DEPC-treated water, and all glassware and spatulas are baked. For pipetting, use disposable plastic.) Linearize the plasmid containing the sequence to be transcribed with a restriction enzyme that cuts downstream of a primer for reverse transcription for example, for...

Polya Tail Analysis

This protocol describes how to label the 3' ends of RNA with cytidine 3',5'- 5'-32P bisphosphate and RNA ligase, followed by hydrolysis of the non-poly(A) portions of the RNA. The remaining labeled poly (A) stretches are separated by electrophoresis and visualized by autoradiography. The method was first developed by Ahlquist and Kaesberg (6) and was later used by Sachs and Davis (7) to demonstrate changes in poly(A) tail lengths in yeast strains deficient in poly(A)-binding protein activity....

Vectors Used for Phage Display

The first phage displaying a protein fragment was made by inserting a foreign sequence into the otherwise wild-type genome of a filamentous phage (Smith, 1985). However, working with phage has a number of disadvantages, such as low yields of DNA and the need to work with plaques instead of colonies. A very popular alternative is to use a phagemid instead. This is a plasmid carrying a filamentous phage origin of replication that allows it to replicate and be packaged in the presence of helper...

Assays Developed To Examine Proteinligand Interactions

During mechanistic studies of the VP55 and VP39 subunits of the vaccinia PAP 2'-O-methyltransferase heterodimer, assays have been developed to study protein interactions with RNA and AdoMet substrates. Some of these are described in the following sections. Several RNA binding assays have been applied to VP55 and VP39. An effective nitrocellulose filter RNA binding assay has been described (Gershon et al, 1991). Application of the electrophoretic mobility shift assay (EMSA) to the analysis of...

Complex Formation A Preparation of Cytoplasmic S100 Extracts

Total cell extracts can be used directly for RNA-protein complex assembly. However, crude extracts contain many sequence-specific and nonspecific RNA-binding proteins. Fractionation of cell extracts may reduce the nonspecific background. For in vitro analysis of RNA-protein interactions thought to occur in the cytoplasm, postnuclear fractions can be prepared by gentle cell lysis and removal of nuclei by low-speed centrifugation. The cytoplasmic fraction can then be further cleared by high-speed...

PREmRna Cleavage And Polyadenylation

Our current understanding of the biochemistry of 3' processing stems, in large part, from the ability to faithfully process exogenously added pre-mRNAs in a reaction dependent on the known cis-acting sequence elements (Hart et al, 1985 Moore and Sharp, 1985 Moore et al, 1986 Sperry and Berget, 1986 Zarkower et al, 1986). An additional advantage of the in vitro system is the ability to uncouple the endonucleolytic cleavage reaction from the poly(A) addition reaction and independently examine the...

Expression And Purification Of The Polya P0lymerase20methyltransferase

The vaccinia PAP subunits are not available commercially but have been expressed and purified on a noncommercial basis. This is relatively straightforward, and a number of options are available, as outlined in the following sections. A. Purification from Vaccinia Virions The vaccinia PAP (VP55-VP39 heterodimer) and cap-specific 2'-0-methyl-transferase (VP39 monomer), like many other vaccinia enzymes, are present within the virion. Until recently, most studies with these enzymes employed...

Alkali Digested RNA Ladder Binding

Prepare the following 20- a1 reaction (in DEPC water) 4 A 5X transcription buffer, 6 A RTP mix (5 A each of 10 mM G, A, U, and C, 40 tl 10 mM DTT), 1 A RNasin (Promega) at 20-40 U fA, linearized DNA template (1 pg DNA usually yields 5-10 fig RNA), and 1 l (15 U) RNA polymerase and incubate at 37 C for 1 hr. Add 1.5 A RQ 1 DNase (Promega) and incubate at 37 C for an additional 15 min. Add 180 jiil 0.3 M sodium acetate, pH 7. Extract with 25 parts phenol 24 parts chloroform 1 part isoamyl alcohol...

Materials and Methods

The fluorescent substrate polyethenoadenosine (poly r(eA) ) was generated by treatment of poly (A) RNA with chloracetaldehyde (48). High-pressure liquid chromatography (HPLC)-purified oligoribonucleotides were purchased from Cruachem and used without further purification. The extinction coefficients (e26o) for each oligoribonucleotide were calculated with the use of the nearest-neighbor approximation method (6). The names, sequences, and e26o values for each of the oligoribonucleotides used in...

Results and Summary

RNA-protein interactions can be evaluated with the use of a number of methods, such as fluorescence, UV absorption, circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy. The only requirement is that the spectroscopic signal, which may be either on the RNA or on the protein, is changed on formation of the RNA-protein complex. The advantage of these methods is their ability to indirectly monitor the steady-state concentrations of the free and bound ligand. In nonequilibrium...

B RNA 3End Labeling

RNA can be 3'-end labeled with a radioactive chain-terminating ATP analog, cordycepin monophosphate (3'-deoxyadenosine-5'-monophosphate or 3'-dAMP). This analog can be transferred to RNA 3' ends using either a recombinant form ofthe PAP encoded by Saccharomyces cerevisiae (Lingner and Keller, 1993) or the heterodimeric vaccinia PAP (Thomson and Gershon, 1995). The vaccinia PAP may provide advantages in terms of its low RNA substrate specificity and ready inactivation. A typical RNA 3'-end...

Extract Preparation

Buffer A 0.3 liter 10 mM N-2-hydroxyethylpiperazine-iV'-2- ethanesulfonic acid HEPES pH 7.9 , 1.5 mM MgCl2, 10 mM KC1, 0.5 mM dithiothreitol DTT Buffer C 0.05 liter 20 mM HEPES pH 7.9 , 25 v v glycerol, 0.42 MNaCl, 1.5 mMMgCl2, 0.2 mM ethylenediaminetetraacetic acid EDTA , 0.5 mM DDT, 0.5 mM phenylmethylsulfonyl fluoride PMSF t Volume discarded cell suspension liters Volume added fresh medium liters This is an example if the cell density is at 5-8 X 105 ml each day before fresh medium is added....