Prokaryotic Translation

Describe the ribosome cycle

• When not translating mRNA, the two subunits can reversibly associate with eachother, dependent on cellular Mg2+ and K+ []

• Physiological conditions strongly favour association

• Dissociation factors bind small subunit to prevent reassociation, these are released during initiation

• Ribosomes 10x more abundant than dissociation factors, only 10% can exist in the dissociated form

Draw a labelled diagram of the ribosome

https://ibb.co/CpmfnYVj

The full process of prokaryotic translation initiation

• 30S associates with IF1 and IF3 (initiation factors)

• 30S:IF1:IF3 associates with mRNA at shine-delgano by 16sRNA bp’ing with SD

• IF2-GTP-citRNA associates, holding citRNA at AUG

• 50S joins

• IF2 intrinsic GTPase activity activated

• Releases all initiation factors

• 70S initiation complex formed

• IF2 exchanges GDP with GTP, recycling

What is the only tRNA that can enter the P site?

• Charged initiator tRNA

• In prok, this is fMet, formyl group rapidly lost by peptide deformylase

• 50% lose rest of Met by slower enzymatic removal

What is the shine-delgano sequence?

• AGGAGG ~10nt upstream of initiator AUG

What evidence is there that the Shine-Delgano site exists?

• Cross link ribosome subunits to mRNA during initiation to prevent elongation and ribosome movement

• Digest unprotected mRNA with RNase

• Isolate and sequence protected fragments

• Locate on genome

• Reveals 16s rRNA binds SD sequence ~ 10nt upstream of initiator AUG

How does the shine-delgano sequence allow for polycistronic translation?

• Ribosome can be bought to each SD sequence individually

• Does not stop translating when next SD sequence is reached

Simply, what is the process of translation elongation?

• EF-Tu brings aminoacyl-tRNA to A site, checks codon-anticodon match and releases, leaving aa-tRNA in A site

• Peptide bond formation

• Translocation transfers new peptide-tRNA to P site

• Deacylated tRNA leaves P site, exits through E site

Draw a detailed diagram showing the process of translation elongation

https://ibb.co/VW65bxdb

What does EF-Tu do?

• Brings aminoacyl-tRNA to A site

• Binds GTP and aa-tRNA, masking aminoacylgroup so cannot react with the currently forming peptide chain

• If codon-anticodon match is correct, conf. change of ribosome triggers intrinsic GTPase activity of EF-Tu, releasing EF-Tu-GDP whilst aa-tRNA remains in A site

Simply, how does prok translation termination occur?

• No tRNAs for termination codons UAG, UAA, UGA

• Release factors recognise termination codons

• Induce hydrolysis of peptide chain from peptidyl-tRNA to release it

• Need action of class I RF, then class II RF

Describe the class I release factors

• RF1, RF2

• Recognise termination codon direclty

• Induce hydrolysis of peptidyl-tRNA at ribsosome

• Released by RF3 GDP-GTP exchange

Describe the class II release factors

• RF3

• Binds ribosome when bound to GDP, exchanges for GTP

• Causes conf. change that releases class I factor

• Subsequent GTP hydrolysis then releases RF3

What is the main way of regulating initiation of translation?

• Accessibility of SD sequence to ribosome

• Binding of trans-acting factors e.g. sRNAs

• Hide SD in thermosensor or riboswitch

Describe the evidence that incorporation of the shine delgano sequence into a hairpin loop inhibits translation

• RNA bacteriophage MS2

• MS2 mRNA has maturase, coat, replicase genes, each with its own SD between each gene, all have potential to form secondary structure

• 21 different mutations made into coat gene that destabilised or stabilised the SD-containing hairpin loop, but did not alter SD or protein sequence

• Translation efficiency measured in vivo]

• Mutations that destabilised the hairpin had increased translational efficiency

• More available SD sequence, more efficient translation

Describe how sRNAs regualte translation? Draw diagrams

• 50-200nt found in ~100 different E.Coli strains

• Bind mRNA

• Can activate by competing with and disrupting secondary structures that are close to and disguising SD sequence

• Can repress by binding directly to SD sequence, preventing IF1:IF3:30S binding and ribosome assembly

• https://ibb.co/HDnt0dg5

How can thermosensors regulate translation?

• Secondary structures that block access to SD (shield or contain)

• Unfold in response to temperature, or the proteins that are synthesised in response to temperature changes e.g. heat shock proteins bind and interfere with stem loop

• Observed in synthesis of prfA protein in some Listeria strains

Describe how Listeria’s prfA acts as a thermosensor

• 5’ UTR forms stem loop that masks SD at 30C (transmission in environment)

• At 37C (infecting host), stem loop disrupted, SD revealed, prfA translated

• prfA increases expression of virulence genes

List 3 main ways of regulating translation

• sRNAs (bind SD)

• Thermosensors / riboswitches (disguise SD)

• Attenuation (prevent mRNA synthesis in the first place)

What is attenuation?

• Regulation of translation by premature termination of transcription (transc. and transl. are coupled in prok).

• Switching off of biosynthetic operons e.g. Trp when corresponding aminoacyl-tRNA is abundant

• Transc. terminating hairpin loop forms in RNA that is still held within RNAP’s exit channel causes dissociation of RNAP

Describe the attenuation of the Trp operon

• Trp operon has 4 ‘regions’

• RNAP transc. first 10-15nt, ribosome recruited behind it (coupled)

• Reaches multiple trp sequence in region 1

• If trp scarce, trp aa-tRNA low, ribosome stalls

• As ribosome can’t reach region 2, 2 can fold and form hairpin, so 2:3 hairpin loop with GCCGC run forms in the mRNA still held within RNAP’s exit channel

• This is handled by TFIIH, transc. and transl. of trp operon continues

• If trp abundant, ribosome does not stall

• Region 2 cannot fold, so instead 3:4 hairpin with UUU run forms in mRNA still held with RNAP’s exit channel

• The rho-independent transc. terminator, RNAP falls off, trp operon not synthesized

Draw and label a simplified representation of a prok. mRNA

https://ibb.co/Tq23C2nh

Extremely simply, how does translation start? How does this link to the majority of prok. translation regulation mechanisms?

• 16s rRNA of 30S binds SD on mRNA

• AUG put in P site

• Regulation determined by SD sequence access, so mechanisms mainly act at 5‘ UTR

What is a riboswitch?

• Complex ds/ss mRNA structure (kind of looks like IRES) that hides SD

• Small molecule linked to metabolism stabilized within the structure

• Different small molecule linked to same metabolic pathway can compete for site and cause new conf. which exposes SD

List 2 cis elements for prok. translation and what they do

• SD - complementary to 16sRNA in 30S to align ribosome with start codon for accurate initiation

• Secondary structures e.g. stem loops can hide SD sequence or IRES to recruit

List 2 trans-acting factors for prok. translation and what they do

• IFs - involved in ribosome initiation complex assembly and proper selection of start codon

• sRNAs - can bind mRNA forming or dissembling hairpin loops for SD disguising / revealing

Draw a diagram showing the full process of translation initiation

https://ibb.co/1G3BkwrH