Nils

Question 1

outline transcriptional regulation in bacteria?

Answer 1

bacterial gene regulation commonly occurs via transcriptional control

activator or repressor binds DNA (positive vs negative regulation)

Question 2

what are the different RNAs in bacteria?

Answer 2

protein-coding e.g. mRNA (make up a small proportion of cellular RNA, most are nc)

non-coding e.g.

• rRNA (16S rRNA in 30s, 5S and 23S in 50S); form complex w ribosomal proteins
• tRNA; transport aa to growing peptide chains,
• regulatory RNAs (not as many as other ncRNAs, but in terms of function are v important)

Question 3

what are regulatory RNAs?

Answer 3

discovered w RNA-mediated reg of ColE1 copy number, abundance of diversity and mechanisms discovered in recent decades

(mostly) small RNAs with diverse mechanisms that control gene expression or protein activity

account for 2-50% of protein-coding genes and control multiple targets

significant regulators in e. coli

Question 4

what are the five categories you can group regulatory RNAs into?

Answer 4

riboswitches

trans-encoded ncRNAs

cis-encoded ncRNAs

dual function RNAs

protein modulating ncRNAs

Question 5

what are riboswitches?

Answer 5

are part of the mRNA they control and regulate; usually located upstream of gene

riboswitch component has two parts; one binds/senses ligand; other controls expression of downstream gene (function)

control of expression may occur through termination, anti-termination, inhibiting or facilitating translation

Question 6

what are trans-encoded regulatory RNAs?

Answer 6

sRNAs located in diff location of genome to the genes they regulate; both get transcribed

work through base pairing interactions with transcripts they regulate; partial complementarity

inhibits or facilitates translation or induces mRNA degradation

Question 7

what are cis-encoded regulatory RNAs?

Answer 7

sRNAs encoded at exact same location as target on opposite strand; complete complementarity

leads to no translation and RNA degradation, mRNA cleavage, or transcription termination

Question 8

what are dual function regulatory RNAs?

Answer 8

sRNAs that have two functions

e.g. SgrS; binds glucose transporter transcript inhibiting translation and causing degradation BUT ALSO encodes small peptide which binds already present glucose transporters preventing more transport - its purpose is to alleviate g6p stress

Question 9

what are protein modulating regulatory RNAs?

Answer 9

sRNAs that bind to and sequester/compete with a protein for its targets

e.g. csrB RNA; in e. coli csrA is global regulator and binds mRNA transcripts via hairpin located at their 5’ end inhibiting translation; csrB forms lots of these hairpins resembling csrA targets; sequesters csrA allowing its targets to be translated

Question 10

what methods are used to discover regulatory RNAs?

Answer 10

three main methods:

computational prediction

direct detection (sequencing or microarray)

functional analysis

(first two only a prediction and/or can’t confirm regulatory function so have to do third to verify)

Question 11

outline discovery of regulatory RNAs?

Answer 11

sometimes accidentally discovered during analysis of a particular process

not traditionally found in genome annotations which focus on ORFs (ncRNAs don’t have)

interest has greatly increased in past decades and they are now known to be highly abundant and widespread

Question 12

how can we predict regulatory RNAs computationally?

Answer 12

impose constraints to eliminate spaces you’re searching in genome; could impose them in this order to narrow down

intergenic regions (many found here)

promoter and terminator search (all RNAs have these; if nc region inbetween could be reg RNA)

size restriction (reg RNAs usually small)

conservation or secondary structure (reg RNAs often conserved in similar species; RNAs w function form particular structure; could expect some base pairing within reg RNA sequence)

to be certain its a reg RNA after all that you need to do functional analysis

Question 13

how can we directly detect ncRNAs with RNAseq?

Answer 13

extract total RNA, do rRNA/ tRNA depletion and size selection to narrow down for small ncRNAs, sequence and map to reference genome

see if any reads mapping to regions w no protein coding genes - could what is being transcribed here is ncRNA

challenges: transcriptional noise (lot of useless transcripts), degradation products, reg RNAs might only be expressed under certain conditions you grew the bacteria in (tedious/costly to test all possible conditions), still don’t know function

Question 14

outline the general mechanism of riboswitches?

Answer 14

they are regulatory elements in mRNA which adopt different conformations in response to signals (e.g. ligand binding, temp change)

two components; aptamer (sensing) and expression platform (responds to sensing)

the altered conformations control expression of the associated gene via termination, anti-termination, no translation or translation

they are not non-coding RNAs but are regulatory RNA sequences

Question 15

outline feedback control in riboswitches?

Answer 15

feedback control is where the end-product of enzymatic pathways regulate its own biosynthesis or transport

riboswitches often control expression of genes encoding a ligand that is recognised by the same riboswitch

Question 16

why is it beneficial that riboswitches are part of the mRNA?

Answer 16

they can interact directly with mRNA allowing immediate control so cell can respond quickly to stimuli

Question 17

outline the importance of riboswitches in regulating gene expression?

Answer 17

widespread in prokaryotes regulating 3% of bacterial genes and also present in archaea and eukaryotes

Question 18

outline the diversity of riboswitches?

Answer 18

can respond to a wide range of molecules including aa’s, metals, cofactors etc.

can form lots of complex structures

Question 19

discuss the role of riboswitches in thiamine biosynthesis?

Answer 19

expression of thiamine biosynthesis genes repressed by thiamine pyrophosphate (TPP) (neg feedback)

5’ UTR of thiM mRNA binds TPP leading to conformational change due to additional basepairing concealing RBS and inhibiting translation

Question 20

how can we study riboswitches?

Answer 20

can generate computer-predicted secondary structures by pasting RNA sequence into online tool which calculates most thermodynamically stable state i.e. how it will fold

some tools even take co-transcriptional folding into account

only a prediction tho and needs to be experimentally proven

Question 21

how can riboswitches be studied experimentally (as opposed to just making a prediction)?

Answer 21

RNA is unstable (cause OH) and spontaneously cleaves; speed of this cleavage dependent on local structure; unstructured regions (e.g. linear) cleave more often/easier

in-line probing involves using RNA probes to examine these patterns in presence or absence of ligand you think causes switch

probe can be purchase or generate through in-vitro transcription; allows you to visualise on gel; you would expect linear region to have bands 1nt apart so this how you determine structure

this how they discovered secondary structure of thiM

Question 22

what are the mechanisms of trans-encoded sRNAs?

Answer 22

incomplete complementarity with target means they can target multiple mRNAs

decrease protein levels through inhibition of translation or mRNA degradation; degradation is by RNaseE and is irreversible

can also activate translation

Question 23

how do trans-encoded sRNAs activate translation?

Answer 23

e.g. DsrA is a trans-encoded reg RNA which activates translation of stationary phase sigma factor RpoS which controls stress response

trans-enc reg RNAs often synthesised under VERY SPECIFIC GROWTH CONDITIONS e.g. oxidative stress, temperature or low iron

DsrA produced under temp. stress; breaks up ds structure which was inhibiting RpoS translation to allow access to RBS; RpoS can be translated and control cellular stress response

Question 24

discuss how trans-encoded regulatory RNAs require Hfq?

Answer 24

Hfq is an RNA chaperone that forms a hexamer mediates contact between regulatory RNA and target; need help due to incomplete complementarity

importance of Hfq in many processes highlighted by its abundance (10,000 copies per cell) and hfq mutants being pleiotropic and any sRNAs in mutant losing regulatory ability

can be used to identify trans-encoded RNAs since they all have Hfq

Question 25

how can we identify trans-encoded sRNAs?

Answer 25

can do Hfq co-immunoprecip and transcriptomics

add tag to Hfq then use this to select for Hfq from all proteins present in cell allowing you to also isolate trans-enc RNAs; sequence alongside wt cells to subtract any non-specifically interacting molecules

then do RNA-seq on all RNAs found and map to reference genome; peaks in Hfq tagged sample that are not in control may have regulatory function

Question 26

discuss the discovery of the trans-encoded RNA MicA?

Answer 26

found MicA through computational search and identified as conserved in many bacteria

used northern blotting (RNA expression) to look at its expression; found it expressed specifically in stationary phase

to identify function they overexpressed MicA on plasmid and did proteomics; showed in 2D page that OmA protein strongly reduced in presence of MicA overexpression