Noncoding RNAs (ncRNAs) have recently been discovered to regulate mRNA transcription in trans, a role traditionally reserved for proteins. The breadth of ncRNAs as transacting transcriptional regulators and the diversity of signals to which they respond are only now becoming recognized. Here we show that human Alu RNA, transcribed from short interspersed elements (SINEs), is a transacting transcriptional repressor during the cellular heat shock response. Alu RNA blocks transcription by binding RNA polymerase II (Pol II) and entering complexes at promoters in vitro and in human cells. Transcriptional repression by Alu RNA involves two loosely structured domains that are modular, a property reminiscent of classical protein transcriptional regulators. Two other SINE RNAs, human scAlu RNA and mouse B1 RNA, also bind Pol II but do not repress transcription in vitro. These studies provide an explanation for why mouse cells harbor two major classes of SINEs, whereas human cells contain only one.

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Summary The short interspersed elements (SINEs) Alu and B2 are retrotransposons that litter the human and mouse genomes, respectively. Given their abundance, the manner in which these elements impact the host genome and what their biological functions might be is of significant interest. Finding that Alu and B2 SINEs are transcribed, both as distinct RNA polymerase III transcripts and as part of RNA polymerase II transcripts, and that these SINE encoded RNAs indeed have biological functions has refuted the historical notion that SINEs are merely “junk DNA.” This article reviews currently known cellular functions of both RNA polymerase II and RNA polymerase III transcribed Alu and B2 RNAs. These RNAs, in different forms, control gene expression by participating in processes as diverse as mRNA transcriptional control, A-to-I editing, nuclear retention, and alternative splicing. Future studies will likely reveal additional contributions of Alu and B2 RNAs as regulators of gene expression.

An undergraduate biochemistry laboratory experiment is described that will teach students the practical and theoretical considerations for measuring the equilibrium dissociation constant (K D ) for a protein/ DNA interaction using electrophoretic mobility shift assays (EMSAs). An EMSA monitors the migration of DNA through a native gel; the DNA migrates more slowly when bound to a protein. To determine a K D the amount of unbound and protein-bound DNA in the gel is measured as the protein concentration increases. By performing this experiment, students will be introduced to making affinity measurements and gain experience in performing quantitative EMSAs. The experiment describes measuring the K D for the interaction between the chimeric protein GAL4-p53 and its DNA recognition site; however, the techniques are adaptable to other DNA binding proteins. In addition, the basic experiment described can be easily expanded to include additional inquiry-driven experimentation.Keywords: Biochemistry, protein/DNA interaction, equilibrium dissociation constant, EMSA.We present an experiment, appropriate for an undergraduate biochemistry laboratory course, that will enable students to measure the equilibrium dissociation constant (K D ) for a protein/DNA interaction using electrophoretic mobility shift assays (EMSAs), also known as gel shifts. This experiment introduces students to the theoretical and practical considerations behind measuring the binding affinity for a biological interaction. Equilibrium binding and dissociation constants are typically introduced in general chemistry; however, their application to biological interactions may not be part of biochemistry curricula. Indeed, many biochemistry students, even at the graduate level, do not firmly grasp these concepts. This experiment provides the opportunity to measure the binding affinity of a protein/DNA interaction.The interaction between the protein GAL4-p53 and its DNA recognition sequence is used as a model system. Protein/nucleic acid interactions are fundamental to all of biology, therefore understanding how to quantitatively measure the affinity of such an interaction, and what that affinity means for biological regulation is important. In addition, this experiment will teach students how to perform EMSAs. This is a basic technique that is widely used in biochemical research laboratories, hence is a valuable technique to learn. Lastly, this experiment provides a framework that can be readily expanded, for example, to teach the relationship between K D and rate constants, or to allow for inquiry-driven experimentation. BACKGROUND AND THEORY

At eukaryotic promoters, multi-faceted protein-protein and protein-DNA interactions can result in synergistic transcriptional activation. NFAT and AP-1 proteins induce interleukin-2 (IL-2) transcription in stimulated T cells, but the contributions of individual members of these activator families to synergistically activating IL-2 transcription is not known. To investigate the combinatorial regulation of IL-2 transcription we tested the ability of different combinations of NFATc2, NFATc1, cJun, and cFos to synergistically activate transcription from the IL-2 promoter. We found that NFATc2 and cJun are exclusive in their ability to synergistically activate human IL-2 transcription. Protein-protein interaction assays revealed that in the absence of DNA, NFATc2, but not NFATc1, bound directly to cJun/cJun dimers, but not to cFos/cJun heterodimers. A region of NFATc2 C-terminal of the DNA binding domain was necessary and sufficient for interaction with cJun in the absence of DNA, and this same region of NFATc2 was required for the synergistic activation of IL-2 transcription in T cells. Moreover, expression of this C-terminal region of NFATc2 specifically repressed the synergistic activation of IL-2 transcription. These studies show that a previously unidentified interaction between human NFATc2 and cJun is necessary for synergistic activation of IL-2 transcription in T cells.