Exploring protein-RNA interactions with site-directed mutagenesis and phage display

The importance of RNA as a mediator of genetic information is widely appreciated. RNA molecules also participate in the regulation of various post-transcriptional activities, such as mRNA splicing, editing, RNA stability and transport. Their regulatory roles for these activities are highly dependent on finely tuned associations with cognate proteins. The RNA recognition motif (RRM) is an ancient RNA binding module that participates in hundreds of essential activities where specific RNA recognition is required. We have applied phage display and site-directed mutagenesis to dissect principles of RRM-controlled RNA recognition. The model systems we are investigating are U1A and CUG-BP1. In this dissertation, the molecular basis of the binding affinity of U1A-RNA beyond individual contacts was investigated. We have identified and evaluated the contributions of the local cooperativity formed by three neighboring residues (Asn15, Asn16 and Glu19) to the stability of the U1A-RNA complex. The localized cooperative network was mapped by double-mutant cycles and explored using phage display. We also showed that a cluster of these residues forms a “hot spot” on the surface of U1A; a single substitution at position 19 with Gln or His can alter the binding properties of U1A to recognize a non-cognate G4U RNA. Finally, we applied a deletion analysis of CUG-BP1 to define the contributions of individual RRMs and RRM combinations to the stability of the complex formed between CUG-BP1 and the GRE sequence. The preliminary results showed RRM3 of CUG-BP1 is a key domain for RNA binding. It possibly binds to the GRE sequence cooperatively with RRM2 of CUG-BP1. RRM1 of CUG-BP1 is not required for GRE recognition, but may be important for maintaining the stability of the full-length CUG-BP1.

Incomplete information games of conflict and collective action

My thesis consists of three essays that investigate strategic interactions between individuals engaging in risky collective action in uncertain environments. The first essay analyzes a broad class of incomplete information coordination games with a wide range of applications in economics and politics. The second essay draws from the general model developed in the first essay to study decisions by individuals of whether to engage in protest/revolution/coup/strike. The final essay explicitly integrates state response to the analysis. The first essay, Coordination Games with Strategic Delegation of Pivotality, exhaustively analyzes a class of binary action, two-player coordination games in which players receive stochastic payoffs only if both players take a ``stochastic-coordination action''. Players receive conditionally-independent noisy private signals about the normally distributed stochastic payoffs. With this structure, each player can exploit the information contained in the other player's action only when he takes the “pivotalizing action”. This feature has two consequences: (1) When the fear of miscoordination is not too large, in order to utilize the other player's information, each player takes the “pivotalizing action” more often than he would based solely on his private information, and (2) best responses feature both strategic complementarities and strategic substitutes, implying that the game is not supermodular nor a typical global game. This class of games has applications in a wide range of economic and political phenomena, including war and peace, protest/revolution/coup/ strike, interest groups lobbying, international trade, and adoption of a new technology. My second essay, Collective Action with Uncertain Payoffs, studies the decision problem of citizens who must decide whether to submit to the status quo or mount a revolution. If they coordinate, they can overthrow the status quo. Otherwise, the status quo is preserved and participants in a failed revolution are punished. Citizens face two types of uncertainty. (a) non-strategic: they are uncertain about the relative payoffs of the status quo and revolution, (b) strategic: they are uncertain about each other's assessments of the relative payoff. I draw on the existing literature and historical evidence to argue that the uncertainty in the payoffs of status quo and revolution is intrinsic in politics. Several counter-intuitive findings emerge: (1) Better communication between citizens can lower the likelihood of revolution. In fact, when the punishment for failed protest is not too harsh and citizens' private knowledge is accurate, then further communication reduces incentives to revolt. (2) Increasing strategic uncertainty can increase the likelihood of revolution attempts, and even the likelihood of successful revolution. In particular, revolt may be more likely when citizens privately obtain information than when they receive information from a common media source. (3) Two dilemmas arise concerning the intensity and frequency of punishment (repression), and the frequency of protest. Punishment Dilemma 1: harsher punishments may increase the probability that punishment is materialized. That is, as the state increases the punishment for dissent, it might also have to punish more dissidents. It is only when the punishment is sufficiently harsh, that harsher punishment reduces the frequency of its application. Punishment Dilemma 1 leads to Punishment Dilemma 2: the frequencies of repression and protest can be positively or negatively correlated depending on the intensity of repression. My third essay, The Repression Puzzle, investigates the relationship between the intensity of grievances and the likelihood of repression. First, I make the observation that the occurrence of state repression is a puzzle. If repression is to succeed, dissidents should not rebel. If it is to fail, the state should concede in order to save the costs of unsuccessful repression. I then propose an explanation for the “repression puzzle” that hinges on information asymmetries between the state and dissidents about the costs of repression to the state, and hence the likelihood of its application by the state. I present a formal model that combines the insights of grievance-based and political process theories to investigate the consequences of this information asymmetry for the dissidents' contentious actions and for the relationship between the magnitude of grievances (formulated here as the extent of inequality) and the likelihood of repression. The main contribution of the paper is to show that this relationship is non-monotone. That is, as the magnitude of grievances increases, the likelihood of repression might decrease. I investigate the relationship between inequality and the likelihood of repression in all country-years from 1981 to 1999. To mitigate specification problem, I estimate the probability of repression using a generalized additive model with thin-plate splines (GAM-TPS). This technique allows for flexible relationship between inequality, the proxy for the costs of repression and revolutions (income per capita), and the likelihood of repression. The empirical evidence support my prediction that the relationship between the magnitude of grievances and the likelihood of repression is non-monotone.