23 510 Finding RNA Sequences That Interact with Other Molecules
wea25324_ch05_075-120.indd Page 114 114 11/10/10 9:48 PM user-f468 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile Chapter 5 / Molecular Tools for Studying Genes and Gene Activity Thus, the protein–protein interactions suggested by such an assay should be verified with a direct assay, such as immunoprecipitation. P T7 SUMMARY Protein–protein interactions can be de- tected in a number of ways, including immunoprecipitation and yeast two-hybrid assay. In the latter technique, three plasmids are introduced into yeast cells. One encodes a hybrid protein composed of protein X and a DNA-binding domain. The second encodes a hybrid protein composed of protein Y and a transcription-activating domain. The third has a promoter-enhancer region linked to a reporter gene such as lacZ. The enhancer interacts with the DNA-binding domain linked to protein X. If proteins X and Y interact, they bring together the two parts of a transcription activator that can activate the reporter gene, giving a product that can catalyze a colorimetric reaction. If X-gal is used, for example, the yeast cells will turn blue. 5.10 Finding RNA Sequences That Interact with Other Molecules DNAs (a) Transcribe (d) PCR cDNAs RNAs (b) Select by binding to a target molecule (c) Reversetranscribe Selected RNAs Figure 5.41 SELEX. Start with a large collection of DNAs (top) that have a random sequence (blue) flanked by constant sequences (red). (a) Transcribe the DNA pool to produce a pool of RNAs that also contain a random sequence flanked by constant sequences. (b) Select for aptamers by affinity chromatography with the target molecule. (c) Reverse-transcribe the selected RNAs to produce a pool of cDNAs. (d) Amplify the cDNAs by PCR, using primers complementary to the constant regions at the ends of the DNAs. This cycle is repeated several times to enrich the aptamers in the pool. SELEX Functional SELEX SELEX (systematic evolution of ligands by exponential enrichment) is a method that was originally developed to discover short RNA sequences (aptamers) that bind to particular molecules. Figure 5.41 illustrates the classical SELEX procedure. One starts with a pool of PCR-amplified synthetic DNAs that have constant end regions (red), but random central regions (blue) that can potentially encode over 1015 different RNA sequences. In the first step, these DNAs are transcribed in vitro, using the phage T7 RNA polymerase, which recognizes the T7 promoter in the upstream constant region of every DNA in the pool. In the next step, the aptamers are selected by affinity chromatography (this chapter), using a resin with the target molecule immobilized. The selected RNAs bind to the resin and then can be released with a solution containing the target molecule. These selected RNAs are then reverse-transcribed to yield double-stranded DNA, which is then subjected to PCR, using primers specific for the DNAs’ constant ends. One round of SELEX yields a population of molecules only partially enriched in aptamers, so the process is repeated several more times to produce a highly enriched population of aptamers. SELEX has been extensively exploited to find the RNA sequences that are contacted by proteins. It is extremely powerful in that it finds a few aptamers among an astronomically high number of starting RNA sequences. Functional SELEX is similar to classical SELEX in that it finds a few “needles” (RNA sequences) in a “haystack” of starting sequences. But instead of finding aptamers that bind to other molecules, it finds RNA sequences that carry out, or make possible, some function. With simple binding, selection is easy; it just requires affinity chromatography. But selection based on function is trickier and requires creativity in designing the selection step. For instance, the first functional SELEX procedures detected a ribozyme (an RNA with enzymatic activity), and this ribozyme activity altered the RNA itself to allow it to be amplified. One simple example is a ribozyme that can add an olignucleotide to its own end. This activity allowed the investigators to supply an oligonucleotide of defined sequence to the ribozyme, which then added this tag to itself. Once tagged, the ribozyme becomes subject to amplification using a PCR primer complementary to the tag. A pool of random RNA sequences may not contain any RNAs with high activity. But that problem can be overcome by carrying out the amplification step under mutagenizing conditions, such that many variants of the mildly active sequences are created. Some of these will probably have greater activity than the original. After several rounds of selection and mutagenesis, RNAs with very strong enzymatic activity can be produced.