RIBOSWITCHES

Abrar AhmedPh. D student, SKIMS

13-03-2013

Riboswitches

Regulatory segment of an mRNA molecule that binds a small molecule (ligand), resulting in a change in production of the proteins encoded by the mRNA.

•conserved genetic control elements in the 5’

untranslated region (UTR) of mRNAs.

• are regulated by metabolites: coenzymes (B1, B2,

B12), amino acids, purine bases.

• consist of only RNA.

• estimation: > 2% of all genes are regulated by

riboswitches.

Organization and properties of riboswitches

Natural aptamers are the basis of riboswitches

Organization and properties of riboswitches

Binding domain (Aptamer):

Regulatory Domain:(Expression platform)

• structured binding pocket

• high affinity:

• high specificity reacts upon ligand binding by the aptamer

comformational change alters gene expression

• Typically provide a feedback mechanism for controlling the expression of metabolic genes

metabolic product of a pathway inhibits expression of proteins required to produce the metabolite.

• RNA domains that modulate gene expression in response to metabolite bindingRiboswitches

Pre-Quinosine-I

2’-deoxyguanosine

Winkler & Breaker (2005) Annu Rev Microbiol 59: 487-517Roth, et al. (2007) Nat Struct Mol Biol. 14: 308-317Kim et al. (2007) Proc. Natl. Acad. Sci. 104: 16092-16097

Known riboswitch aptamers

Known riboswitch aptamers

• RFN element in mRNAs of genes required for biosynthesis of riboflavin and FMN

• FMN required for repression of the ribD operon of B. subtilis

• Mutations in the RFN element eliminate FMN-mediated repression of the operon

Regulation of riboflavin biosynthesis in B. subtilis

•Ronald Breaker et al. used in-line probing technique to prove

that RFN element is an aptamer that binds directly to FMN.

•In-line probing: The efficient hydrolysis of a phosphodiester bond

in RNA needs a 180 degree (“in-line”) arrangement among the

attacking nucliophile (water), the phosphorus atom in the

phosphodiester bond, and the leaving hydroxyl group at the end

of one of the RNA fragments created by the hydrolysis.

RNA analysis by in-line probing

ribD mRNA

The RFN element is a riboswitchRonald R Breaker (2002), PNAS 99, 15908-13

How riboswitches regulate gene expression?

Gene regulation by riboswitches:

-Transcription termination (FMN riboswitch)

-Translation inhibition (thiamine riboswitch)

- RNA cleavage (glmS riboswitch)

Transcription termination

Repression

Activation

ON OFF

The FMN riboswitch terminates transcription

Translation inhibition

Repression

Activation

ON OFF

thi operon of B. subtilis

RNA cleavage

Tom Cech, Nature (2004) 428: 263-264.

Cleavage of RNA by a riboswitchWinkler and Dann (2006), Nat Struct Mol Biol 13, 569-71

Summary• regulatory elements which control a wide set of metabolic pathways in

bacteria (mostly).

• consist solely of RNA

• Aptamer domain: high affinity and specific binding of metabolites

• Expression platform: regulation of gene expression by control of

- transcription termination

- translation initiation

- RNA stability

SIGNIFICANCE OF RIBOSWITCHES

Barrick & Breaker (2006) Scientific American 296: 50-57

Thanks