lecture 2: riboswitches

what are riboswitches

segments of an mRNA that can directly bind to a specific small molecule and dictates whether the mRNA is translated or destroyed, found in 5’UTR of mRNAs

aptamer domain of a riboswitch

receotir that binds to a specific ligand

expression platform of a riboswitch

has mutually exclusive conformations, allowing expression (or not)

switching sequence of a riboswitch

part of the aptamer or expression platform depending on ligand binding

• when bound to metabolite, it is base-paired with the aptamer

• when metabolite is not bound, the switching sequence binds to a different base downstream

riboswitch attenuation mechanisms

1. metabolite binding

2. tRNA binding

metabolite binding (e.g. Sam-I riboswitch)

SAM is a precursor for making methionine and cysteine. the bacteria only wants to spend energy making these amino acids when the levels are too low

• low SAM: the mRNA forms an antiterminator loop, which allows RNA polymerase to keep moving past the regulatory region and finish transcribing the entire genome

• high SAME: SAM binds to the RNA, causing massive re-shuffling of RNA’s shape, forcing a terminator loop which physically yanks the RNA polymerase off the DNA before it reaches the gene

tRNA binding (e.g. T-box riboswitches)

the T-box riboswitch binds to tRNA, allowing the cell to measure how many amino acids are currently available for building proteins

• high amino acid levels: most tRNAs are loaded so the riboswtich does not bind these tRNAs efficiently. the mRNA folds into a terminator shape and the cell stops making enzymes

• low amino acid levels: tRNAs are unloaded so they bind to the T-box riboswitch on the mRNA, stabilising the antiterminator loop, allowing for transcription

the shine-dalgano sequence

a short purine-rich sequence, located just upstream of the start codon

when does occlusion occur

when the SD sequence is physcially blocked/ hidden, preventing the ribosome from binding

occlusion of the SD sequence (e.g. TPP)

TPP is the active form of vitamin B1 and is a vital cofactor that enzymes need to handle carbon metabolism

• low TPP: no TPP is bound to the aptamer so the expression platform stays in a configuration that keeps the SD sequence exposed, so the ribosome can easily find it

• high TPP: TPP binds to the aptamer, forcing the expression platform to change its 3D shape, making the SD sequence occluded and physically blocks the ribosome

ribozymes (e.g. the GlmS ribozyme)

this ribozyme controls the enpression of GlmS, which is involved in the synthesis of glucosamine-6-phosphate

• low GclN6P: the GlmS mRNA remains intact annd stable so the ribosome can bind

• high GclN6P: GclN6P binds to the GmlS mRNA, triggers the RNA to self-cleave. the mRNA is now broken into 2 pieces and ribonucleases recognise these broken ends and quickly degrades them

mechanisms by which regulatory RNA affects the expression of prokaryotic genes:

• transcription interference

• attenuation

• blocking the SD sequence

• regulating the activity of endonucleases

what regulates riboswitches

small RNAs

antisense RNA

single-stranded RNA molecules that are complementary to sense mRNAs

transcription interference example

expression of mccA from C. acetobutylicum

MccA is an enzyme involved in the conversion of methionine to cysteine and is required when Met lelvels are too high but cys levels are too low

T-box

regulates a promoter that transcribes genes in a forward direction (senses when cysteine is low)

S-box

regulates a promoter that transcribes an antisense transcript in the opposite direction (senses when methionine is low)

what happens when both cysteine and methionine are low?

both the T-box and S-box allows their RNA polymerase to start moving. because they are on the same piece of DNA, the 2 RNA polymerases collide. when they hit each other, they both stall and fall off the DNA

neither transcript is finished so no enzymes are produced. by allowing this, the cell can prioitise the conservation of methionine

transcription attenuation (similar to the riboswitch mechanism)

binding of an antisense RNA promotes the formation of a termination stem loop

• high levels of metabolite: expression of sRNAs stabilise the termination stem loops so no expression of metabolism genes

• low levels of metabolite: no expression of sRNAs so an anti-termination stem loop forms and allows for expression of the metabolic genes

substrate for nuclease altered (e.g. stophylococcus aureus RNAIII)

RNA III is expressed in response to a high level of infection and regulates a large number if virulence factors like SPA macrophages. there are 2 mechanisms:

• RNA III is complementary to the hairpin of the SPA mRNA, blocking the SD sequence and start codon

• RNAse III is a ribonuclease that cleaves dsRNA. RNAIII binds to mRNA targets RNAse III. the mRNA is cleaced within the ds region then fully degraded by other nucleases

indirect translation blocking

the sRNA binds to a part of the mRNA that is not the SD sequence, forcing a conformational change in the mRNA. the mRNA folds over itself, hiding the SD sequence

direct translation blocking

an sRNA is produced that is complementary to the SD sequence. because the SD sequence is now double-stranded and occupied, the ribosome cannot recognise/ bind to it

give an example of direct translation blocking

micF sRNA regulates expression of OmpF porins.

high osmolarity/ toxins produces various transcriptional regulators which activates micF, causing OmpF translation to be inhibited so less nutrients and less toxins enter the cell

RNA thermometers

a highly sensitive, temperature-responsive sequence found in the 5’UTR of the mRNA. they allow bacteria to sense changes in temperature and immediately adjust their protein production without any helper proteins

what is the primary function of RNA thermometers

to control the accessibility of the SD sequence

phase variation

a survival strategy used by bacteria to rapidly and reversibly switch expression of certain genes on or off

slipped-strand mispairing (phase variation) and give an example

• causes addition/ deletion of repeats, causing a frame shift leading to a non-functional protein/ premature stop

• e.g. H. pylori: Slip stranded mispairing of a CT dinucleotide within the ORF leads to premature stop codons, allowing it to escape the immune system

methylation (phase variation) in E.coli

E.coli has 2 methylases: Dam and Dcm

• replication of methylated DNA leads to hemi-methylated daughters.

• replication of hemi-methylated daughters leads to 1 methylated and 1 unmethylated strand

• proteins binding to sites overlapping the methylation sites can block access of the methylase and stabilise the unmethylated state

what can methylation block and give an example of this

blocks regulatory proteins that can only interact with unmethylated DNA, adding a degree of randomness to protein binding

• e.g. E.coli P fimbriae sticks to host tissues, specifically during UTIs

• in the off state: LRP binds to sites 1,2 and 3, blocking the promoter for the pap gene and prevents it from methylation

• in the on state: LRP shifts and binds to sites 4,5 and 6 so the promoter is free

invertable genetic switches (phase variation)

• high frequency genetic switches that can control the expression of nearby genes

• the segment of DNA that is going to be flipped is bound by inverted repeats. to flip the DNA, the 2 ends must physically touch (uses NAPs) to bend the DNA)

• a specific recombinase recognises the repeats and cuts the DNA at both repeat sites, rotating it 180 degrees and religates the DNA

give examples of ivertible genetic switches

1. E.coli type I fimbriae

2. C.difficile CwpV