RIBOSWITCHES

GENETIC CONTROL ELEMENTS

Introduction Identifying the first riboswitches Proofs of roboswitches Occurance Molecular structure Regulation by various mechanisms Examples in prokaryote and Eukaryote Tools and software RNA world Applications and Prospects

INTRODUCTION

Genetic control is an essential feature of living system, as cells must respond to environment and many biochemical signals by varying genetic expression patterns. Most known mechanisms of genetic control involve use of protein factors that sense chemical and physical stimuli and then modulate the gene expression by selectively interacting with the DNA/ mRNA

Riboswitch is coined by Dr. Ronald Breaker in 2002 when he reported that mRNA encoding enzymes of vitamin B1 and B12 biosynthesis in E.coli could bind associated metabolites (without helper proteins) and they are RNA based components that integrate ligand binding and gene regulation to respond to molecular signals within cells.

IDENTIFYING THE FIRST RIBOSWITCHES

It is known that expression of genes responsible for synthesis and import of coenzyme B12 in bacteria is repressed when the levels of this essential coenzyme were adequate. Several attempts were made to identify the protein factor responsible for sensing coenzyme B12 and repressing these genes, no evidence of such a protein was found.

Same is the case in riboflavin mediated control for biosynthesis of FMN and also thiamine pyrophosphate biosynthesis. These unexplainable gene control mysteries that were first examined for possible riboswitch function.

PROOF OF RIBOSWITCHES

Four publications in 2002 provided the evidence to confirm mysteries involved in the action of metabolite binding RNAs

A combination of RNA probing and equilibrium dialysis assays confirmed that the most highly conserved portions of the 5UTRs of the suspected mRNAs indeed form highly specific receptors that dock with their target metabolites in the complete absence of proteins Site directed mutagenesis experiments and gene expression assays invivo confirmed that metabolite binding by these RNAs was critical for proper gene control.

ribD operon in B.subtilis control synthesis and transport of vitamin riboflavin and its product FMN. Bacterial rib operon contain conserved sequence at 5 UTR known as RFN element. Mutation in this region abolish normal control of ribD operon in FMN. Hypothesis- RFN interacts with protein that respond to FMN or FMN itself. To test the hypothesis that FMN directly binds to the aptamer Ronald Breaker team used a technique called in-line probing.

IN-LINE PROBING

Efficient hydrolysis is possible when RNA is unstructured whereas RNA constrained by secondary structure or by binding to the ligand cannot. Therefore hydrolysis occurs in unstructured and linear RNA easily than with secondary structure or ligand.

Gel electrophoresis results of inline probing Lane 1= no RNA Lane 2= RNA cut with Rnase T1 Lane 3= RNA cut with base Lane 4 & 5 = spontaneous cleavage in absence and presence of FMN for 40 h at 25OC. Arrows denote regions of RNA that became less susceptible to cleavage in the presence of FMN.
The changes in susceptibility were half maximal at FMN concentration at 5nM. This indicates high affinity between RNA and ligand.

OCCURANCE

Riboswitches occur in bacteria, but functional riboswitches of one type ( the TPP riboswitch) have been discovered in plants and certain fungi. TPP riboswitches have also been predicted in archaea, but have not been experimentally tested. In 2009 the riboswitch was discovered in humans

STRUCTURE

1) 2) 3)

A typical mRNA transcript consists of three sections

5 UnTranslated region (5UTR) Protein coding region (from start codon to stop codon) 3 UnTranslated region (3 UTR)

MOLECULAR ARCHITECTURE

1.

2.

Riboswitches need to form molecular architectures with sufficient complexity to carryout two main functions Molecular recognition: Aptamer senses a single ligand (structured binding pocket, high affinity, high specificity) Regulatory Domain (Expression platform) : controls gene expression (reacts upon ligand binding by the aptamer, conformational change alters gene expression)

MOLECULAR RECOGNITION

Riboswitches are structured noncoding RNA domains that selectively bind metabolites and control gene expression (Mandal and Breaker 2004a; Coppins et al. 2007; Roth and Breaker
2009)

CONFORMATIONAL CHANGES ALTER GENE EXPRESSION

When metabolite is not bound (-M), the expression platform incorporates the switching sequence into an anti-terminator stem loop (AT), and the transcription proceeds through the coding region of the mRNA. When metabolite binds (+M), the switching sequence is incorporated into the aptamer domain, and the expression platform folds into a terminator stem-loop (T), causing transcription to abort.

REGULATION

Riboswitches regulate several metabolic pathways Biosynthesis of vitamins (e.g. riboflavin, thiamine and cobalamin & Metabolism of methionine , lysine and purines. Riboswitches regulates at transcription initiation, post transcriptional regulation, translation initiation, and a variety of other functions.
Recently CRISPR RNAs (clustered regularly interspaced short palindromic repeats) have been shown to form a basic adaptive immune system of bacterial cells by interfering with plasmid and phage infections.

VARIOUS MECHANISMS ADOPTED BY RIBOSWITCHES

RIBOSWITCH MECHANISM

RIBOSWITCH

Transcription termination

RIBOSWITCH

Translation initiation

RIBOSWITCH

Ribozyme mediated RNA cleavage

ILLUSTRATION OF APTAZYME BASED RIBOSWITCH SENSOR

RIBOSWITCH ACTING AS A RIBOZYME

Studied in B.subtilis. glms gene Codes for the enzyme glutamine fructose-6-phosphate amido transferase. Enzyme induces conformational change in the leader sequence and activates ribozyme Cleavage of the leader region occurs 245 nucleotides upstream of the AUG codon Leads to reduced expression Cleavage enhanced by 1000 fold in the presence of GlcN6P

The B.subtilis glmS riboswitch is conserved upstream of the glmS gene. During conditions of excess GlcN6P, the glmS 5 UTR is stimulated to self cleave at a specific site at its 5 end. Cleavage leads to glmS repression through an unknown mechanism

RIBOSWITCH

RNA splicing control

IVS intervening sequence

Riboswitches in plants
Metabolite mediated control of splicing and

alternative 3 end processing of mRNA transcripts.

RIBOSWITCHES IN NEUROSPORA CROSSA

RIBOSWITCHES
6S RNA : the structure of 6S RNA is highly conserved among bacteria and was hypothesized to be a key for RNAP binding as it resembles a DNA open promoter. Transcription of 6S RNAdependent genes by the housekeeping RNAP (exponential phase) is inhibited by large amounts of 6S RNA under nutrient limitation (stationery phase). Upon resupply with nutrients (outgrowth), RNAP is released by the synthesis of short

CATEGORIZATION OF RIBOSWITCHES

Aptamer domains are used to distinguish each known class of riboswitch because they remain extraordinarily well conserved in sequence and secondary structure, even among distant related organism. According to existing database there are 10 main riboswitch classes, these riboswitches sense a variety of ligands (metabolites). In contrast to the highly conserved aptamer domains expression platforms can vary widely in primary sequence and in mechanism of action.

RIBOSWITCH STRUCTURES

TOOLS AND SOFTWARE

Riboswitch finder: analyze a sequence using web interface and then checks specific sequence elements and secondary structure calculates and displays the energy folding of the RNA structure. (www.biozentrum.uniwuerzburg.de/bioinformatik/riboswitch/.) RibEx (Riboswitch Explorer): searches any sequence for known riboswiches and for other predicted ones. (www.ibt.unam.mx/biocomputo/ribex.html.) CMfinder (Covariance model based RNA motif finding algorithm): this tool performs well on unaligned sequences and also when the motif is only present in a subset of sequences. Used for motif search, structure predication combing folding energy and sequence co-variation. (http://wingless.cs.washington.edu/htbinpost/unrestricted/CMfinderWeb/CMfinderInput.pl.) RiboSW: tool for searching putative riboswitches in a sequence. The tool had two parts, the RNA secondary structure and functional region to search putative riboswitches. http://ribosw.mbc.nctu.edu.tw/.

RIBOSWITCHES ARE ANCIENT

Certainly the presence of riboswitch like structures would have enhanced the efficiency of metabolic processes in organisms of the RNA world. Obviously these first riboswitches would not have been controlling the expression of protein enzymes, but they would have controlled the production, processing and activity of their ribozyme counterparts. There is possibility that the riboswitches discovered are close relatives of RNA world metabolite sensors. This could be a fact that cells need to sense the concentrations of important compounds and that RNA has been chosen for this task.

EMERGING APPLICATIONS OF RIBOSWITCHES

Riboswitches as tools for regulated gene expression. Riboswitches as antimicrobial targets. Riboswitch-based control of bacterial behaviour.

Riboswitches as tools for regulated gene expression

Ligand inducible expression systems are important genetic tools in any common laboratory. Inducers (such as IPTG) are too expensive to be useful on industrial scale. Natural riboswitches that are activated by amino acids may therefore represent an affordable alternative for such applications. Tandem glycine riboswitch form B.subtilis was used for glycine inducible production of Beta galactosidase which provide only 6 fold induction when glycine was added.

STUDYING MICROBIAL GENETICS

Ligand sensitive riboswitches are useful in production of conditional hypermorphic mutants, particuarly in case where null mutants are lethal. Example: theophylline sensitive synthetic riboswitch was designed to regulate an essential gene that modulate motility (csrA). The creation of csrA-lacZ fusion at this chromosomal position verified that expression levels were low in absence of theophylline but approached wild type expression levels in the presence of theophylline. The cells were non-motile in absence of inducer but regained their motility in presence of theophylline. This experiments showed that csrA is a negative regulator of autoaggregation in E.coli.

RIBOSWITCHES AS ANTI-MICROBIAL TARGETS

Riboswitches could be a target for novel antibiotics. Indeed, some antibiotics whose mechanism of action was unknown for decades have been shown to operate by targeting riboswitches. Ex: Pyrithiamine enters cell, it is metabolised into pyrithiamine pyrophosphate. Pyrithiamine pyrophosphate has been shown to bind and activate the TPP riboswitch, causing the cell to cease the synthesis and import of TPP. Because pyrithiamine pyrophosphate does not substitute for TPP as a coenzyme, the cell dies.

RIBOSWITCHES BASED CONTROL OF BACTERIAL BEHAVIOR

Bacterial chemotaxis has been studied extensively and well understood at genetic level. The ability to modulate bacterial motility in response to arbitrary chemical signals would provide new tools for bioremediation and drug delivery Example: cheZ chemotaxis signaling protein is reprogrammed and placed under the control of theophylline sensitive switch. Reprogrammed cells would then migrate up gradient of this ligand and autonomously localized to regions of high theophylline concentration, which is a behaviour that cannot be accomplished by natural E.Coli chemotaxis.

Riboswitches can be applied as biosensors, metabolic engineering of organisms and in gene therapy treatments. It has shown the potential to act as a drug target against various bacterial diseases. Riboswitches turn on and off depending on the Concentration of the ligand. Antibacterial strategy would be to design metabolite mimics that bind on a riboswitch receptor, shut off expression of the adjoining genes, and starve the cell of important metabolites generating analogues of the ligands that bind to riboswitches and regulate key metabolic pathways may become a new approach for developing antimicrobial agents. THE END