LIFE AND TIMES OF AN RNA, L4/5 MCDOWALL

THE LIFE OF RNA IN E.COLI – BACTERIAL MODEL

BACKROUND KNOWLEDGE AND THEN REQUIRES BACKGROUND READING – sub-evaluation of

experimental work – carry on also read references on his slides.

Describe mechanisms by which RNase E controls fate of cellular RNAs in bacteria? - Many type of
mechanism – 5’monophophate sensing, further regulation of interaction between RNse E and RNA
control factors, spatial temporal regulation and cellular fate of RNA – switch from translation to
degrading (all occurs in cytoplasm so how is this controlled)

RNAase E

 Rnase e is regulator in degradation and is hypothesised to be localised

within inner surface of membrane – spatial organisation
 Endonuclease enzyme within a degradosome complex

mRNA DEGRADATION/ TURNOVER/ DECAY

 Key in the central dogma – RNA equally important and is regulated,

RNA that is not function id localised to the ribosome to be translated
 Mrna is unstable hence to be maintained in the cell it has to be
continually transcribed – hence translation follows closely programs
of transcription – otherwise it would rapidly be degraded.
 mRNA instability is key in the regulation of gene expression as well,
RNA degradation is regulated at many levels.
 Program response of degradation pathway to control transcription
 Cycle balance – hence degradation is key in recycling of nucleotides for transcription.
 Rna polymerase, ribosomes and RNAse E complexes in Mrna processing.
 Manipulation of mrna degradation can be used to change overall gene expression – Jacobson,
peltz PTC therapeutics (cystic fibrosis)
 Therapeutics for inhibitors of RNase E – antimicrobials
 MODEL FOR DEGRADATION – e.coli; RNA is degraded by endonucleases and 5’-3’ exonucleases
combination but NO 3’-5’ exonuclease activity.
- Full length mRNA with stem loop structure at 3’ end and poly
adenylated tail after – oligoadenylated tails; destabilise the mrna
accelerating rate of decay
- Eukaryotes – once initiated, exonucleases stall at stem loop – hence
addition of poly a tail and allow additional contacts to degrade stem
loop structure.
 Co-transcriptional translation occurs in e.coli and not in eukaryotes (where ribosomes are
starting on the mRNA which Is still being transcribed (shown in electron micrograph) with
polyribosomal complexes – rates of transcritpional and translation very similar
- With transcription, 5’ end of mRNA may become increasing separated from nucleoid – hence
spatial separation within the cell is important in the process
 mRNA could be degraded entirely by 3’-5’ exonucleases – so what do endonucleases do?
- Offer an alternative to Mrna being degraded from 3’ end – as clashing between 5-3 ribosomes
and 3-5 exonucleases may occur, wasteful mechanism also – hence RNAse E minimises this clash
as endonuclease – cleaves predominantly behind translating ribosomes
- Accelerate rate of turnover of mRNAs – once initiated – by generating 3’ ends that are more
accessible to 3’-5’ exonucleases – nascent 3’ ends of bacteria have a stem-loop – feature a part
of the transcription termination signal; rho dependant tor rho independent – hence impede
exonucleases, hence endonucleases allow intercut to allow access of exonucleases – degradation
occurs faster as more cutting points?
- Process polycistonic (multiple coding segments on mrna) mrna into segments that have different
rates of degradation; not every gene is expressed at same level in final product from single
mrna, endonucleases cleavage polycistonic RNA with upstream and downstream stability affects
degradation.
- By-product of needing endonucleases for the maturation of non-coding RNAs (rrna and trna)
- Provide a means of co-degradational, co-transcriptional translation - Clear stalled ribosome is
premature termination occurs (mutation) allows cleavage of ribosome or if a block in translation
– sense upstream signals on mrna.

RNASE E MECHANISM & DEGRADATION

 One of the multiple endonuclease in the cell – rnase e –

essential as temperature sensitive mutant/ activation results in
stabilisation of many transcripts in E.coli – loses activity at high
temperature hence allows to determine effects –
 Role in processing of precursors of small, non-coding RNAs as
well as stable RNAs
 Magnesium dependant endoribonuclease conserved across
bacteria – antimicrobial? Also found in chloroplasts
 Single strand specific cleavage at A or U or both rick sequences, in E.coli RNase E is physically
associated with other factors involved in RNA degradation – RNA degradosome
 DEGRADOSOME – reduction approach found catalysis and specificity determined by the NTH (n-
terminal half activity) of the RNase E – evolutionary conserved domain. Also defined minimal
substrates
 Found that RNase E is homotetramer, the NTH is soluble where the tetramer binds one to four
synthetic oligonucleotide substrates – multiple contacts with RNA stabilising interaction as its a
tetramer (4 points of contact - cooperativity), co- crystal structure shows dimer of dimers.
 S1 domain – fold to allow interaction with RNA (found in many RNA binding proteins), catalytic
activity – Dnase family. S1 claps RNA onto DNase 1 domain (closed conformation) where open
conformation, the s1 domain swings open – flexible structure.
 Terminal phosphate at 5’end of oligonucleotide – 5’ monophosphate. Where the rnase E has
5’sesnsing pocket. 2 amino cids contact monophosphate in hydrogen bonding – thr170 and
arg169 contacts monophosphate 5’ end recognition.
- Terminal phosphate can be locked into place by semi-circular ring of h bonds by
thr 170 & arg169
- Interaction of the arg169 is consolidated by gyl124
- Terminal base forms hydrophobic contacts with the side chain val128
- Triphosphorlyted group is occluded (doesn’t fit) nor ds segment hence
5’recongition
- Pocket was predicted – RNASE E cleavage of some substrates enhanced by 5’
ends that terminate in a few unpair nucleotides and monophosphate – first
endonuclease reported that can sense 5’ rna phosphorylation state – suggesting that 5’ sensing
ensured once degradation is initiated, it goes to completion
 When RNase cleaves it creates down stream monophosphate nucleotide
(products) – rnase is stimulated by mono-phosphorylated end hence
downstream product is preferred substrate for rnase e. all or none – resent on
western blot – all rna disappears (MACKIE)
 Forms of substrates with base pairing at 5’ end or were circular were less
susceptible to rate limiting cleavages by RNase E (BELASCO & MACKIE)
 Ds regions don’t fit in pocket – ds segment to 5’ of mRNA (STEMLOOP) is
stabilises the mRNA, tri-phosphorylated end doesn’t fit either. The mrna is
unrecognised by rnase E but also the stem loop prevents interact – stabilising
mrna – hence how are mono-phosphorylated mrna created – susceptible to
RNAase E?
- RNA pyrophosphodyrolase – removes 2 phosphates from tri-phosphorylated
ends – mrnas involved in longevity – stem loop structure still protects against this – similar to
decapping in eukaryotes

5’ MONOPHOSPHORLYATED END-DEPENDENT PATHWAY

 RppH – pyrophohydrolase (decapping enyme) – cleaves the 2

phosphates to get mono – cleavage by RNAse which is then
produces another substrate and then endonucleases can then chop
up the rest.
 Recognition mechanism
 5’ stem loop hinders decapping as well as interaction with 5’
monophosphate (stabilises mRNA), hence inactivation of RppH
stabilises many mRNA transcripts also. CALAGHAN ET AL 2005
 Rnase E and 5’ MPEDP offers a pathway by which clashes between ribosomes (5-3) and
exonucleases (3-5) are minimised – assuming initial cleavage blocks translation initiation and
then subsequent cleavage occurs in the 5’-3’ direction behind translating ribosomes.
 Functional coupling with 3’-5’ exonuclease ensures rapid removal of translation initiation signals
and other segments to which ribosomes would otherwise bind non-productively
- Ribosomal subunits interact with rna, ribosome binding site is required for translational
positioning – due to splicing mechanisms and exonuclease - many rnas are produced, hence
ribosomes have to sort through all rnas for shine delgarno sequence
- To avoid this, functional coupling between exonuclease so degradation intermediates don’t
accumulate in the cell as this would reduce translation efficiency due to high RNA intermediates.
– as the degradosome contains an RNA helicase, rnase E and PNPase 3-5’ exonuclease
 Central dogma – dominant mechanism

RECRUITMENT/ ACTIVATION OF RNase

 RNase can be stimulated by small 5’monophosphorlyted RNA – antisense RNA that bins mrna
mediate downregulation of gene expression by bringing monophosphate to the mRNA – hence
cell can change gene expression by mono-phosphorylated end
 Degradation machinery – inner surface of membrane hence transcription starts in nucleotide
and translation in cytoplasm – spatial organisation – allow ribosomes to interact with mRNA
before the degradosome hence allows preference for ribosome
 Ribosomes have high affinity for rnas which do not contain a shine delgarno sequence –
ribosomes don’t search – predominant place for initiation Is the 5’UTR (untranslated region)
 Found invitro that RNase E cleaves RNA without the presence of 5’ monophosphate – KIME ET
AL 2014 – rapid cleavage of RNA by RNase E In absence of 5’ monophosphate stimulation –
oligonucleotides had 3Gs at 5’ end (RNA using T7 polymerase) hence non-WC base pairing
occurred causing a ring of 4 oligonucleotides through G interaction – causing tails on
nucleotides. Single stranded regions of in the quadruplex can fir in active site on RNase E –
through recognition of multiple single stranded regions. – not accepted yet.
- Hence rnase interacts with multiple single stranded regions – hence doesn’t require 5’
monophosphate – multivalent interactions (multiple interacts give specificity and affinity).
Activate of rhhp -results in stabilisation of 100s transcripts – hence may be another mechanism

Adjacent single stranded regions mediate processing of tRNA precursors by RNase E direct entry.

 Trna contains antiparallel stem loop structures and RNase is found in processing of tRNA –
structural very stable also hence easier to work with than mRNA – allows easier detection of the
effects of mutation.
 Processing of trna – long precursor is cleaved using RNase E and further processed with endo
and exo nucleases.
- Transcription of mrna with mono or tri phosphorylated ends
- Or can mutate RNase E – t170v mutation – blocks cleavage of mono-phosphorylated nucleotides
but still cleavage of5’ mono-hydroxylated ends
 Not 5’ end dependant – what is it dependant on? Block sites of single stranded regions with
primers or deletion of sites where rnase e may bind
- Cleavage sites, E3 and E5; removal of E5 site – lose e3 also and vice versa. Wt substrate get
cleavage at E3 OR E5 but not both.
- Hence RNASE E binds ss region and cleaves one – loose
interactions after and falls off (ie multiple footholes in the wall
hence doesn’t require a handle ie monophosphate)
- Occurs alongside monophophate dependant patwhay
 Hence another mechanism: DIRECT ENTRY
 Shown using trna precursors – biochemical evidence that direct entry
cleavage of natural transcripts is mediated by specific unaired regions
that are adjacent to but not contiguous with segments cleavage by
RNase E
 Confirms invivo evidence that processing of trna in E.coli is not dependant on interaction with
5mp, hence does direct entry have significant role beyond trna precursors – mrna included
- T170v mutant used again (blocks sensing of monophos end hence has to use direct entry) this
was used with tri-phosphorylated ends also to prevent
monophos cleavage
- samples of e.coli RNA was depleted of 23s and 16s Rrna and
incubated with NTH-RNase E and T170V rnase – RNA
components of e.coli – monophos was removed and was
treated with hydroxylase to give 5’ hydroxyl groups – ONLY
DIRECT ENTRY hence dependency of cleavages on interaction
with 5’-monophoslplhyted end was investigated using
sample of monophos or hydroxylated 5’ends
- Positions of rnae e cleavage were then mapped – rna
sequencing approach. 5’ adapter to 5’end of rna species
before sequencing – downstream products and new 5’ends
 are generated using adapters allowing identification of rnase cleavage – biochemcial studies,
clarke and kime et al 2014
 Direct entry is major pathway of mRNA degradation – direct entry on endonuclease to cleavage
internally without interaction with 5’monopohphate – hence direct entry by rnase e is key in
mrna degradation in e.coli
- Identified 1000s of sites for which the corresponding downstream products increased when
T170V was incubated with 5’hyroddxyklated RNA invitro and decreased following inactivation of
RNasee E invivo.
- Hallmark of RNase E – ability to interact with 5’ mono-phosphorylated ends – not required for
efficient cleavage at plethora of sites within the e.coli transcriptome – mrna, trna rna etc.
- Analogous enzymes in other bacteria – see similar mechanisms from rnases in these

SPECULATION

 When mra is first produced – 5’UTR is first made – hence rnase E is capable of interacting with
this region – can also now interact with single stranded regions hence competition of binding
between rnase e and the ribosome at the initiation step mostly. – initial competeion between
rnase and ribosomes at level of translation nintiation via shared interaction with 5’UTR
 By following behind rna polymerase, ribosomes are unlikely to provide opportunity for rnase to
cleave in front of it
 Further from nucleotide, the greater ability of degradosome to bind. RNASE E binds 5’UTR and
can block ribosome binding – naked mrna hence other rnase E can bind and cleave – ordered
transition from translation to degradation
 Surveillance mechanism – defective mrna – stalled ribosomes – rnase E can act by direct entry
 Rnase E – dynamic molecule in open conformation, binds many substrates and completely fit
hence in closed conformation, brings S1 domain onto site of catalysis – binding without cleavage
 Rnase e can bind stem loop regions – important in rnase E being recruited by small antisense
rnas – increased rates of degradation by increased mono-phosphorylated ends but also can do it
by binding stem loop structures so rnase e can bind and interact.

BLCOKING TRANSALTION

 RNASEe binds and physically recruits it to the inner

membrane – octopus tentacles – pulls out from rna
pool – increased spatial recognition
 Rnnase e binding to translation initiation regions
will not only block the binding of ribosomes but
recruit mrna to degradosome complexes
 With passing of the last ribosome – regions internal
to coding regions become exposed to
endonucleolytic cleavage hence fragments are
degraded by 3’ exoRNases – removal of key
intermediates and allows sources of translation
machinery to be reused – hence major source of
nucleotide recycling.
 Glycolytic enzyme – enolase in degradosome –
speculated it sense nutritional status of cell and
feedback to degradome to control rate of mrna
turnover – slow down protein synthesis

KEY POINTS FOR EXAM