Regulatory RNAs

• Regulation by RNAs in Bacteria(sRNA, Riboswitches &

CRISPR)
• Regulatory RNAs are Widespread in Eukaryotes
• Synthesis and Function of miRNA and siRNA Molecule
• Silencing Gene Expression by Small RNAs
• Long Non-Coding RNAs(ncRNA) and X-Inactivation
 Regulation by RNAs in Prokaryotes
• RNA molecules that act as regulators were known in bacteria for years before the first microRNAs (miRNAs) and
short interfering RNAs (siRNAs) were discovered in eukaryotes.

• RNA regulators in bacteria are a heterogeneous group of molecules that act by various mechanisms to modulate
a wide range of physiological responses.

• Regulatory RNA in prokaryotes include:

1. sRNA - (Trans-acting): The largest and most extensively studied set of small RNA (sRNA) regulators acts
through base pairing with RNAs, usually modulating the translation and stability of mRNAs. The majority
of these small RNAs regulate responses to changes in environmental conditions.
6S RNA, RybB, Dsr A, Rpr A, Oxy S

2. Riboswitches (Cis-acting): These are part of the mRNAs that they regulate. These leader sequences
fold into structures amenable to conformational changes upon the binding of small molecules.
Riboswitches thus sense and respond to the availability of various nutrients in the cell.

3. CRISPR: Finally, a recently discovered group of RNA regulators, known as the CRISPR RNAs, (Clustered
Regularly Interspaced Short Palindromic Repeats) contains short regions of homology to bacteriophage
and plasmid sequences. CRISPR RNAs interfere with bacteriophage infection and plasmid conjugation,
most likely by targeting the homologous foreign DNA through an unknown mechanism.
 sRNA (Trans-acting)
•Small regulatory RNA genes have been found in most bacterial species for many years.

•sRNA transcripts are generally less than 200 nt (80 - 100 nucleotides).

•Some are involved in regulating the replication of plasmids, and others are involved in regulating gene expression.

•sRNAs can control gene expression at the level of translation or transcription.

•sRNAs respond to environmental or internal stress conditions.

•sRNAs are not generally formed by processing of larger double-stranded RNA (dsRNA) precursors (as those eukaryotic
RNA regulators –miRNA & siRNA); instead, they are encoded in their final form by small genes.

•They are, however, larger (80–110 nucleotides) than those eukaryotic regulatory RNAs (which range from 21 to 30
nucleotides).

•More than 100 sRNAs being uncovered in E. coli.

•sRNAs generally form small and imperfect RNA/RNA base pairing with target mRNA, directing destruction of the target
mRNA, inhibiting its translation or even in some cases stimulating translation.
 6S RNA- Shift in Expression to 𝛔S Promoters
• The bacterial 6S RNA was the first sRNA shown to inhibit transcription by binding directly to the housekeeping
holoenzyme form of RNA polymerase (i.e. 𝛔70 -RNA polymerase in E. coli).

• 6S RNA binds to the 𝛔70 subunit of RNA polymerase and down-regulates transcription from many 𝛔70 promoters.

• The 6S RNA accumulates at high levels in stationary phase (the growth- phase in which bacteria enter as
nutrients become depleted and the cells stop dividing).

• In stationary phase, an alternative 𝛔 factor, 𝛔S, is made. This 𝛔S competes with 𝛔70 for core polymerase and
directs the enzyme to promoters expressing genes for the multiple stress responses needed to survive
stationary phase.

• By downregulating transcription from 𝛔70 promoters, 6S RNA helps this shift in expression to 𝛔S promoters.

• Binding of an sRNA to its target mRNA is in most cases aided by the bacterial RNA chaperone protein Hfq which
is needed because the complementarity between the sRNAs and their target mRNAs is typically imperfect and
short, and thus their interaction is weak. Hfq facilitiates base pairing. Also, by binding the sRNAs even before
they are paired with their targets, Hfq increases the stability of these regulators.
 Lets Study Some More sRNAs
• A well-studied sRNA from E. coli is the 81-nucleotide RybB RNA. This sRNA binds several target mRNAs and
triggers their destruction because the double-strand stretch of heteroduplex formed upon pairing is recognized
as a substrate by the nuclease RNase E. Most of the mRNAs targeted by RybB encode iron storage proteins.
RybB regulates the levels of free iron by controlling the levels of iron storage proteins.

• The stationary-phase 𝛔 factor 𝛔S is encoded by the rpoS gene of E. coli. Translation of rpoS mRNA is stimulated
by two sRNAs: DsrA and RprA. Activation is achieved by a switch in alternative RNA base pairing: the small RNAs
bind to a region of the mRNA that otherwise would pair with the ribosome-binding site, inhibiting translation.
The rpoS gene is also acted on negatively by another small RNA, OxyS.

• RBS is occluded by base pairing with another region of the same RNA molecule - translation is inhibited.

Activators DsrA and RprA bind to a region of the mRNA that otherwise
would pair with the RBS thereby Activating Translation.
• RBS is occluded by base pairing with another RNA molecule - translation is inhibited.

Repressor OxyS bind to RBS, occluding RBS thereby Inhibiting Translation

 Riboswitches (Cis-acting regulatory RNA in prokaryotes)
Perhaps the simplest bacterial RNA regulatory elements are sequences at the 5′ end of mRNAs that can adopt different
conformations in response to environmental signals, including stalled ribosomes, uncharged tRNAs, elevated temperatures,
or small molecule ligands.
Structure of Riboswitch

Riboswitch

Responds to Ligand Responds to Uncharged tRNA

Binding

• Riboswitches generally consist of two parts:

Transcription Attenuation Aptamer region, which binds the ligand, and the
Termination Loop RBS Occluded in Trp so-called Expression platform, which regulates
Followed by U’s gene expression through alternative RNA
structures that affect transcription or translation.
• The aptamer binds the controlling metabolite,
causing changes in the structure of the adjoining
Transcription No Initiation of expression platform.
Termination Translation
 Riboswitches Can Regulate Transcription Termination
Numbers 1 – 4 indicate different sequence elements within the RNA upstream of the
coding region (yellow). In the absence of SAM, regions 2 and 3 form a stem-loop; in the
presence of SAM, regions 1 and 2 form a stem-loop, and regions 3 and 4 do likewise. The
consequence of that change in secondary structure controls transcription or translation as
shown.

A stem-loop of regions 3 and 4 is followed by a stretch of Us in the mRNA, which triggers RNA polymerase to terminate
transcription immediately after transcribing those regions and before entering the downstream coding region. This form of
transcriptional regulation is also called Attenuation.
 Riboswitches Can Regulate Translation Initiation

The stem-loop formed by regions 3 and 4 inhibits translation initiation by

sequestering the ribosome-binding site.
Riboswitches Respond to a Range of Metabolites
 The trp Operon

• The 20 common amino acids are required in large amount for protein synthesis,
and E. coli can synthesize all of them.

• The genes for the enzymes needed to synthesize a given amino acid are
generally clustered in an operon and are expressed whenever existing supplies
of that amino acid are inadequate for cellular requirements.

• When the amino acid is abundant the biosynthetic enzymes are not needed and
the operon is repressed.
 Regulatory Sequence

This operon is regulated by two mechanisms:

1. The repressor binds to its operator

2. The transcription of trp operon is attenuated.

trp Operon in the Absence of the Co-Repressor
trp Operon in the Presence of the Co-Repressor
 Amino Acid Biosynthetic Operons Are Controlled by Attenuation
The trp operon is controlled by repression and attenuation, providing a two-stage response to progressively more stringent
tryptophan starvation. But attenuation alone can provide robust regulation: other amino acid operons such as leu and his
rely entirely on attenuation for their control. In the case of the leucine operon, its leader peptide has four adjacent leucine
codons, and the histidine operon leader peptide has seven histidine codons in a row.

The trp operon- The tryptophan operon of E. coli, showing the relationship of the leader to the structural genes that code
for the Trp enzymes. The gene products are anthranilate synthetase ( product of trpE), phosphoribosyl anthranilate trans-
ferase (trpD), phosphoribosyl anthranilate isomerase-indole glycerol phosphate synthetase (trpC), tryptophan synthetase b
(trpB), and tryptophan synthetase a (trpA).
 trp operator leader RNA- Features of the nucleotide sequence of the trp
operon leader RNA.
The sequence of the 5’ end of the trp operon mRNA includes a 161-nucleotide leader sequence
upstream of the first codon of trpE. Near the end of this leader sequence, and before trpE, is a
transcription terminator, composed of a characteristic hairpin loop in the RNA (made from sequences
in regions 3 and 4), followed by eight uridine residues.
Transcription Termination at the trp Attenuator
Transcription termination at the trp operon
attenuator is controlled by the availability of
tryptophan.

(a) Conditions of high tryptophan: sequence

can pair with sequence 4 to form the
transcription termination hairpin.

(b) Conditions of low tryptophan: the ribosome

stalls at adjacent tryptophan codons, leaving
sequence 2 free to pair with sequence 3, thereby
preventing formation of the 3 – 4 termination
hairpin.

(c) No protein synthesis: if no ribosome begins

translation of the leader peptide AUG, the hairpin
forms by pairing of sequences 1 and 2, preventing
formation of the 2–3 hairpin, and allowing
formation of the hairpin at sequences 3–4. The
Trp enzymes are not expressed.
 CRISPR RNAs
• Before we move on to consider the role of regulatory RNAs in eukaryotes, there is one
more system to consider in bacteria. Although it is not strictly an example of gene
regulation (it is a system of defence against viruses and other extrachromosomal
intruders-Plasmids), the mechanism used is strikingly similar to systems we will
encounter in eukaryotes, namely, RNAi.

• CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA

sequences found in the genome of prokaryotic organisms such as bacteria.

• These short palindromic repeats are highly conserved within a given cluster separated by
unique spacer that are non-identical DNA that matches perfectly with bacteriophage
DNAs or plasmids. This quickly led to the proposal that these arrays are involved in some
sort of defence mechanism against foreign nucleic acids entering the cell.

• Each crRNA contains 8 nucleotides of the 5’ repeat followed by the complete spacer
region and most of the next repeat. The repeat sections included in the crRNA are called
the 5’ and 3’ handles, respectively, and represent conserved parts of every crRNA.
CRISPRs Are a Record of Infections Survived and Resistance Gained
• Each crRNA contains 8 nucleotides of the 5’ repeat followed by the complete spacer region and most of the
next repeat. The repeat sections included in the crRNA are called the 5’ and 3’ handles, respectively, and
represent conserved parts of every crRNA.

Unique spacer sequences

Short segment (20 – 40

nucleotide long) of repeated
palindromic DNA sequence
form Hairpin structure
 The Organization of the CRISPR Locus

The conserved repeat sequences and variable spacer sequences are shown at the top.
Underneath is an array of such sequences (the number varies enormously); the proximal
leader sequence is also shown. The generic features consist of repeated sequences (each
approx. 30 bp long and highly conserved within a given cluster) interleaved with spacer
sequences of similar length but highly divergent sequence. At one end of the array is a so-
called leader sequence, often A-T rich and approx. 500 bp in length.
 The CRISPR-Cas System is a Prokaryotic Immune System
• They also identified CRISPR associated genes- cas genes - A set of conserved protein-coding genes tightly associated with
the CRISPR sequences.
• These cas genes are going to make cas proteins that include helicases and nucleases. Together this is called CRISPR-Cas
complex.
• The unique spacer sequences are derived from DNA fragments of bacteriophage that had previously infected the
prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence
these sequences play a key role in the antiviral (i.e. anti-phage) defence system of prokaryotes.
• The CRISPR-Cas system confers bacterial resistance to foreign genetic elements such as those present
within plasmid and phages. RNA harbouring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and
cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign DNA. CRISPR are found in approximately 50% of
sequenced bacterial genome.

CRISPR-Cas Complex
 The Organization of cas-Genes at Three CRISPR Loci

A set of conserved protein-coding genes is tightly associated with the CRISPR sequences.
The two most highly conserved members (cas1 and cas2, for “CRISPR associated”) are
found in all cases, but other cas-genes, and more distantly conserved genes, are less so.
These genes encode proteins involved in different aspects of CRISPR function.
 The Antiviral Operation of the CRISPR
• When the bacteriophage infects the cell, the cell transcribe and translate these cas-protein of the CRISPR-complex that
include the helicase and nuclease and also transcribe this RNA –crRNA that matches the phage DNA.
• This complex includes one sub- unit that is implicated in the processing of the long transcript into the individual short
crRNAs, each the length of one spacer and one repeat sequence. These small RNAs remain bound to the Cascade
complex and direct it to the DNA genomes of invading foreign DNA
• This cr-RNA will fit into the cas-protein complex. It will pair with the phage DNA and will break it, killing the phage before
it can infect the cell. Hence acting as an immune system of the bacteria.
 CRISPR - Infection with a New Virus
• What happens if we have an infection where the CRISPR system doesn't have the matching DNA?
• The system this time will activate a different class of cas-protein that make a copy of this DNA and insert into the
CRISPR complex as spacer DNA.
• Hence - What CRISPR actually is - Spacer-Repeat-Spacer-Repeat where spacer is the history of past infection of
the bacteria, so that the bacterial cell wouldn't be infected again.
• Hence, DNA sequences derived from foreign DNA ( phage or plasmids) are accumulated in regions of the genome
that can subsequently be expressed in the form of small RNA molecules that destroy homologous nucleic acids
should they invade the cell again.

The mechanism of spacer sequence acquisition

Watch this video on CRISPR Technology

https://youtu.be/MnYppmstxIs