Genetika Molekuler (7)

1. Genetika Molekuler (7) Sutarno

2. Lecture #5 Notes (Yeast Genetics) • LECTURE 5: THE TWO-HYBRID SYSTEM AND SUPPRESSORS • The techniques that we’ve been going over are used to study the function of individual genes. But in the cell proteins have to function together, either as parts of complexes, or in pathways where they respond to other genes/proteins. This is what we are now going to move on to: genetic techniques that we can use to find and study interacting genes and getting clues to how they interact with each other. • If you’ve got a gene, and you’re not sure exactly what it is doing, one big clue that can help is to find out what proteins it interacts with. You may still not know what it does, but if you can find out what it does it to (or with), you’re in much better shape. • Finding interactors • Biochemically • Co-IP • Co-purify • Affinity columns • Far-western • Genetically • Other mutants with the same phenotype (the more specific the phenotype, the stronger the connection) • Two-hybrid system • Genetic modifiers (suppressors and synthetic lethals) • THE TWO-HYBRID SYSTEM • A very widely used genetic method for uncovering physical interactions. • It was first devised in yeast, using a transcription-based assay, but the great thing about it is that it is not yeast–specific, but can be used to detect interactions between proteins from any species. • And despite the fact that the most common version uses a transcription-based assay for detecting interactions between two proteins, it is not restricted to studying transcription factors (in fact, it presents additional problems when studying transcription factors). • First described in 1989 (Fields and Song Nature 340: 245-246) • The experimental basis for the two-hybrid system • Since activators are DB proteins, the first question was whether DB activity was sufficient for activation. • Activator proteins have separable domains (DBD and AD) • Each domain by itself is insufficient to activate transcription. • ADs can be fused to heterologous DB domains (therefore, DBD recruits the AD) • Some transcription factors recruit the AD non-covalently, through protein-protein interactions • Fields realized that this can be used as an assay to detect protein-protein interactions in other proteins. • He tested if the idea would work, using two proteins (Snf1 and Snf4) that were already know to physically interact using other methods. • Three components to the system: (jargon of the system) • DBD fusion (usually Gal4DBD or LexA DBD) (Gal4DBD-Snf1 in the original test) (=bait) • AD fusion (usually Gal4 AD) (Snf4-Gal4 AD in the original) (=prey) • Reporter genes (usually LacZ or HIS3, with appropriate binding sites for the DBD upstream) • The hoped-for results: OffOn • Bait fusion alone White His- • Prey fusion alone White His- • Bait + Prey Blue His+ • It worked! (Bait and prey fusions alone were each insufficient; activation required both parts of each fusion protein, as predicted) • This now allows you to ask the question: Does protein X interact with protein Y by making specific fusions of each and testing them in this system. • BUT it can also be used to ask a much more powerful (and more broad) question: What proteins interact with protein X? (due to improvements in the system) • Improvements: • His+ selection • AD cDNA library (the 2000 Clontech catalog listed 69 different libraries) • Getting a positive signal in the two-hybrid assay does not mean that the two proteins interact in vivo…it is a great way to identify CANDIDATES, but they have to be tested in other ways. • A problem: There is a high background of false positives, and also false negatives (not all interactions will be detected). Attempts have been made to eliminate those problems (new systems), to improve the specificity without losing sensitivity, but it makes it especially important to perform controls. • Controls/steps to test if interaction is real: • Is the detected interaction specific? • Does the purified plasmid re-transform the 2-hybrid interaction? • Does it activate by itself or with the DBD? (require the bait fusion)? • Does it interact with non-specific baits? (lamins) • Is it biologically relevant? • Do they co-localize (immunolocalization / cell fractionation) • Biochemically  Do they interact by other assays (co-IP, GST pulldowns) • Genetically  Reverse genetics (related mutant phenotypes? Overexpression suppress? • Double mutant phenotypes?) • Many derivatives of the two-hybrid system have been devised (not clear to me how • frequently they are successful) • One-hybrid (DNA-protein interactions) • Three-hybrid (RNA-protein interactions) • Reverse two hybrid (blocking/inhibiting known interactions) (especially useful in the pharmaceutical industry) • E. coli and pol III versions • Non-transcriptional derivatives (Sos system) • Comprehensive directed genomic 2-hyb arrays have been somewhat disappointing (see Hazbun and Fields, PNAS 98:4277-4278 (2001) for their evaluation…little overlap between attempts, and only ~13% of the known interactions detected) • GENETIC MODIFIERS • Another way to identify other proteins/genes that have a function related to that of YFG is to look for mutations that modify the phenotype of an existing mutation. • Two types: • Suppressors  results in a phenotype more like wild type • Enhancers  results in a more severe phenotype (the most severe phenotype is death, so a common class of enhancers identified in yeast is synthetic lethals) • The reason this works: the starting mutation can sensitize the pathway. • The phenotype doesn’t have to be complete (partial suppression or synthetic sickness), but just has to be reproducible enough to follow the responsible mutations. • In addition to identifying new genes and making new connections between genes, suppression can be used to order the components of a pathway. (see Hinnebusch and Fink for a great example) • Absolutely routine to look for suppressors in yeast…any strong decent sized genetic lab is likely to be doing a suppressor hunt of some type. • Finding and analyzing suppressors/enhancers occurs essentially the same as any other mutant hunt • Dominant / recessive test • Due to a single mutation? • Complementation tests • Linkage analysis • Cloning • Other tests have to be done to help make sense of suppressors (see below) • Finding suppressors establishes a genetic connection/link between two genes. But then the question becomes what is the molecular basis for that connection? (why does one mutation suppress the other?) To address that question we need to know the types of mechanisms that cause suppression, and then we can go over how to distinguish between those possible mechanisms. • TYPES / KINDS / MECHANISMS OF SUPPRESSION • Intragenic • A second mutation in another part of the gene restores function (compensates for the original mutation) • A mutation at the same codon, but to a different amino acid • GAA  Glu (WT) UAA  STOP (original mutation) UUA  Leu (more like WT phenotype) • Restore the wild type sequence (true revertant) • This class might be useful for structure/function studies, but generally this is not the class that most people look for when they set up a suppressor hunt • no new gene has been identified • no new relationship between genes • no pathway • Informational(alter the passage of information encoded in the DNA to protein) • tRNAs (nonsense and frameshift suppressors) • components of the translation apparatus • usually you want to avoid these too…unless you happen to be studying tRNAs, frameshift suppression, or translation. But if you want to get them, there are ways to more specifically get them (by starting with a nonsense allele, etc.) • Amount • Alters the amount of the mutant protein, by a variety of mechanisms • Promoter mutation • RNA processing machinery • More stable RNA or protein • Duplication of the mutant gene • Transcription factor mutation • Might provide information about how the gene / protein levels are regulated, but not necessarily about its function • Activity • Alters the activity of the mutant protein • Some intragenic mutations • Removal of direct inhibitors • Mutation in directly interacting protein, restoring the interaction • Although not necessarily by the lock-and-key mechanism • (a la Allison Adams with actin and Sac6, where interactions in different parts of the protein are strengthened) • mutual suppression is a good indication of direct interaction • post-translational modifications • Same pathway • Mutations in one part of a pathway compensate for defects in another part of the pathway • (similar to the activity class, but increasing the pathway, not by increasing the activity of the original mutant protein) • increase the activity of other pathway components • removal of inhibitors of the pathway • counteract parts of the pathway (kinase mutation suppressed by a phosphatase mutation) • Different pathway • Bypassing the need or reliance for the original pathway (parallel pathway) • Another protein acquires the function of the original protein • Examples: E. coli maltose permease mutant is suppressed by a lactose permease mutation that now transports maltose • hip1 (histidine permease) or mup1 (methionine uptake) suppressed by gap1 (general amino acid permease)