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Synthetic gene networks can be used to control gene expression and cellular phenotypes in a variety of applications. In many instances, however, such networks can behave unreliably due to gene expression noise. Accordingly, there is a need to develop systematic means to tune gene expression noise, so that it can be suppressed in some cases and harnessed in others, e.g. in cellular differentiation to create population-wide heterogeneity. Here, we present a method for controlling noise in synthetic eukaryotic gene expression systems, utilizing reduction of noise levels by TATA box mutations and noise propagation in transcriptional cascades. Specifically, we introduce TATA box mutations into promoters driving TetR expression and show that these mutations can be used to effectively tune the noise of a target gene while decoupling it from the mean, with negligible effects on the dynamic range and basal expression. We apply mathematical and computational modeling to explain the experimentally observed effects of TATA box mutations. This work, which highlights some important aspects of noise propagation in gene regulatory cascades, has practical implications for implementing gene expression control in synthetic gene networks.

Although several recent studies have focused on gene autoregulation, the effects of negative feedback (NF) on gene expression are not fully understood. Our purpose here was to determine how the strength of NF regulation affects the characteristics of gene expression in yeast cells harboring chromosomally integrated transcriptional cascades that consist of the yEGFP reporter controlled by ( i ) the constitutively expressed tetracycline repressor TetR or ( ii ) TetR repressing its own expression. Reporter gene expression in the cascade without feedback showed a steep (sigmoidal) dose–response and a wide, nearly bimodal yEGFP distribution, giving rise to a noise peak at intermediate levels of induction. We developed computational models that reproduced the steep dose–response and the noise peak and predicted that negative autoregulation changes reporter expression from bimodal to unimodal and transforms the dose–response from sigmoidal to linear. Prompted by these predictions, we constructed a “linearizer” circuit by adding TetR autoregulation to our original cascade and observed a massive (7-fold) reduction of noise at intermediate induction and linearization of dose–response before saturation. A simple mathematical argument explained these findings and indicated that linearization is highly robust to parameter variations. These findings have important implications for gene expression control in eukaryotic cells, including the design of synthetic expression systems.

Students design primers using web-based tools along with a printed sequence of a prokaryotic gene showing both DNA strands and translation of the coding strand into amino acids. The melting temperature of each primer is determined and the pair closest in temperature can be used to amplify and clone a gene into a vector for a one-semester laboratory exercise. By working through this primer design exercise, students see first hand what is involved in the process of amplifying DNA.

Background: Transcription factors (TFs) play a central role in regulating gene expression by interacting with cis-regulatory DNA elements associated with their target genes. Recent surveys have examined the DNA binding specificities of most Saccharomyces cerevisiae TFs, but a comprehensive evaluation of their data has been lacking. Results: We analyzed in vitro and in vivo TF-DNA binding data reported in previous large-scale studies to generate a comprehensive, curated resource of DNA binding specificity data for all characterized S. cerevisiae TFs. Our collection comprises DNA binding site motifs and comprehensive in vitro DNA binding specificity data for all possible 8-bp sequences. Investigation of the DNA binding specificities within the basic leucine zipper (bZIP) and VHT1 regulator (VHR) TF families revealed unexpected plasticity in TF-DNA recognition: intriguingly, the VHR TFs, newly characterized by protein binding microarrays in this study, recognize bZIP-like DNA motifs, while the bZIP TF Hac1 recognizes a motif highly similar to the canonical E-box motif of basic helix-loop-helix (bHLH) TFs. We identified several TFs with distinct primary and secondary motifs, which might be associated with different regulatory functions. Finally, integrated analysis of in vivo TF binding data with protein binding microarray data lends further support for indirect DNA binding in vivo by sequence-specific TFs. Conclusions: The comprehensive data in this curated collection allow for more accurate analyses of regulatory TF-DNA interactions, in-depth structural studies of TF-DNA specificity determinants, and future experimental investigations of the TFs' predicted target genes and regulatory roles., National Human Genome Research Institute (U.S.) (grant R01 HG003420), National Human Genome Research Institute (U.S.) (grant R01 HG003985), American Heart Association (postdoctoral fellowship 10POST3650060)