Cyclic di-GMP is a circular RNA dinucleotide that functions as a second messenger in diverse species of bacteria to trigger wide-ranging physiological changes, including cell differentiation, conversion between motile and biofilm lifestyles, and virulence gene expression. However, the mechanisms used by cyclic di-GMP to regulate gene expression have remained a mystery. We demonstrate that cyclic di-GMP in many bacterial species is sensed by a riboswitch class in mRNA that controls the expression of genes involved in numerous fundamental cellular processes. A variety of cyclic di-GMP regulons are revealed, including some riboswitches associated with virulence gene expression, pili formation, and flagellar organelle biosynthesis. In addition, sequences matching the consensus for cyclic di-GMP riboswitches are present in the genome of a bacteriophage.
Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used 2 approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs, to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly expressed mRNAs contained a highly structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide variety of primary sequences. Using a set of eGFP mRNAs with independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e., mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability.
Riboswitches are structured RNA domains that can bind directly to specific ligands and regulate gene expression. These RNA elements are located most commonly within the noncoding regions of bacterial mRNAs, although representatives of one riboswitch class have been discovered in organisms from all three domains of life. In several Gram positive species of bacteria, riboswitches that selectively recognize guanine regulate the expression of genes involved in purine biosynthesis and transport. Because these genes are involved in fundamental metabolic pathways in certain bacterial pathogens, guanine-binding riboswitches may be targets for the development of novel antibacterial compounds. To explore this possibility, the atomic-resolution structure of a guanine riboswitch aptamer from Bacillus subtilis was used to guide the design of several riboswitch-compatible guanine analogs. The ability of these compounds to be bound by the riboswitch and repress bacterial growth was examined. Many of these rationally designed compounds are bound by a guanine riboswitch aptamer in vitro with affinities comparable to that of the natural ligand, and several also inhibit bacterial growth. We found that one of these antimicrobial guanine analogs (6-N-hydroxylaminopurine, or G7) represses expression of a reporter gene controlled by a guanine riboswitch in B. subtilis, suggesting it may inhibit bacterial growth by triggering guanine riboswitch action. These studies demonstrate the utility of a three-dimensional structure model of a natural aptamer to design ligand analogs that target riboswitches. This approach also could be implemented to design antibacterial compounds that specifically target other riboswitch classes.
Many genes elicit their actions through their expression in precise spatial patterns in tissues. Photoregulated expression systems offer a means to remotely pattern gene expression in tissues. Using currently available photopatterning methods, gene expression is only transient. Herein is described a general method to permanently alter a cell's genome under the control of light. The photocaged estrogen receptor (ER) antagonists, nitroveratryl-hydroxytamoxifen (Nv-HTam) and nitroveratryl-hydroxytamoxifen aziridine (Nv-HTaz), mediate exposure-dependent recombination in cells expressing the Cre-ER, a fusion of the site-specific recombinase Cre and ER. Both Nv-HTam and Nv-HTaz only activate recombination by Cre-ER after exposure to light. When released only intracellularly, the covalent-modifying Taz can mediate significant amounts of recombination in an exposure-dependent manner. Nv-HTaz and Cre-ER represent perhaps the first compound that can be used to photopattern gene expression through recombination.
Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used two approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly-expressed mRNAs contained a highlystructured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide-variety of primary sequences. Using a set of eGFP mRNAs that independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e. mRNA being actively translated).
In the past two decades, remarkable advances have been made in the development of technologies used to engineer new aptamers and ribozymes. This has encouraged interest among researchers who seek to create new types of gene control systems that can be made to respond specifically to small molecule signals. Validation that RNA molecules can exhibit the characteristics needed to serve as precision genetic switches has come from the discovery of numerous classes of natural ligand-sensing RNAs called riboswitches. While a great deal of progress has been made towards engineering useful designer riboswitches, considerable advances are needed before the performance characteristics of these RNAs match those of protein systems that have been co-opted to regulate gene expression. In this review, we will evaluate the potential for engineered RNAs to regulate gene expression and lay out possible paths to designer riboswitches based upon currently available technologies. Furthermore, we will discuss some technical advances that would empower RNA engineers who seek to make routine the production of designer riboswitches that can function in eukaryotes.
Full-length hammerhead ribozymes were subjected to in vitro selection to identify variants that are allosterically regulated by theophylline in the presence of a physiologically relevant concentration of Mg(2+). The population of allosteric ribozymes resulting from 15 rounds of in vitro selection yielded variants with observed rate constants (k (obs)) as high as 8 min(-1) in the presence of theophylline and maximal k (obs) increases of up to 285-fold compared to rate constants measured in the absence of effector. The selected ribozymes have kinetic characteristics that are predicted to be sufficient for cellular gene control applications, but do not exhibit any activity in reporter gene assays. The inability of the engineered RNAs to control gene expression suggests that the in vitro and in vivo folding pathways of the RNAs are different. These results provide several key pieces of information that will aid in future efforts to engineer allosteric ribozymes for gene control applications.
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