Chapter 7: small RNAs and riboswitches

function of RNA

serves as: information storage (mRNA), enzyme (ribosome), component of CRISPR (interference and maturing sgRNA) and regulator (asRNA and riboswitches)

Why are antisense RNAs (asRNA) much shorter than the mRNA molecules to which they bind?

asRNA is 15-50nt long. Only pairing of 6-12 nucleotides near the RBS is enough to inhibit translation

How do cells synthesis antisense RNA molecules?

cells do NOT produce asRNA. A distinct anti-gene encodes for asRNA

What happens to mRNA molecules following binding of their antisense RNAs?

asRNA binds to the complemented mRNA and forms dsRNA. This cannot be translated and will be degraded by specific ribonucleases

What are riboswitches?

small molecules sensing units at 5'-end of mRNA, regulating gene expression when product or substrate is present

What happens when a riboswitch binds the small molecule it recognizes?

When bind it can turn transcription or translation ON/OFF

function of riboswitch - Aptamer

binds to metabolite (substrate of product)

function of riboswitch - expression platform

leads to changes in mRNA expression

Anti-SD sequence

part of the 30S ribosome, uses Anti-SD to bind to the mRNA and positions it into the right way to start the translation at the start codon.

SD sequence

Shine-Delgarno sequence. Upstream of the startcodon of the gene

where start translation if riboswitches are involved?

translation does not begin at the 5' end if riboswitches are around

short explanation riboswitch model - transcription

in absence of ligand, premature terminator is present, transcription stops

in presence of ligand, premature terminator is absent, transcription continues

short explanation riboswitch model - translation

in absence of ligand, SD is blocked by anti-SD, translation stops

in presence of ligand, SD is free, anti-SD:anti-anti SD are bound, transcription continues

Riboswitch transcription - activating

binding of substrate to riboswitch removes the terminator from mRNA being produced, RNA-polymerase continues transcribing

Riboswitch transcription - repressing

binding of product to riboswitch causes conformation change of terminator, RNA-polymerase stops

Riboswitch translation - activating

binding of substrate to riboswitch makes anti-anti RBS bind to anti-RBS, creating a hairpin. RBS is available and the ribosome can tanslate

Riboswitch translation - repressing

binding of product to riboswitch causes binding of anti-RBS to RBS, the SD sequence gets included in a stem-loop. Ribosome can't bind, translation is blocked

Boolean logic gates

illustrates the dynamics of 2 riboswitches (AND/ NAND/OR/NOR)

input: presence of metabolite

output: expression of a gene

What does SELEX stand for?

Systematic Evolution of Ligands by EXponential enrichment

explain the SELEX procedure

A library of variants is screened for candidates that binds to a target bound by a bead. Binders are isolated, the target is removed. Binders are amplified using RT-PCR

What is SELEX?

method for isolation of strong binding RNA/DNA against a target

alternative uses of riboswitch (pharma)

as an antibiotic, by mimicking the structures of co-factors

Antisense RNA only inhibits....

Translation

Riboswitches controls....

Transcription and Translation

ribosome binding site (RBS)

few nucleotides upstream the startcodon, A and G rich region

What does the transcription terminator consist of?

hairpin loop with a poly U sequence

tmRNA

tRNA and mRNA hybrid that is used to free stalled ribosomes