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

• IF2 binds GTP and charged initiator tRNA

• Associates with complex already at SD, holding citRNA at P site

• 50S joins

• IF2 intrinsic GTPase activity activated

• GTP hydrolysis releases all initiation factors

• IF2 exchanges GDP with GTP, recycling

• 70S initiation complex formed

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 by transfer of the polypeptide being formed from P site to the aa-tRNA in A site

• Translocation transfers new peptide-tRNA to P site

• Deacylated tRNA leaves P site, exits through E site

Simply, what are the three elongation factors required for prok translation elongation?

• EF-Tu

• EF-G

• EF-T

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

• Joins A site

• 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

What does EF-G do?

• Helps translocate ribosome

What does EF-T do?

• Helps recycle EF-Tu from GDP to GTP bound

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 or by presence of upstream RNA secondary structures, which can be further influences by translation of other ORFs or temperature (thermosensors)

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) can be sensitive 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

• At 37C, stem loop disrupted, SD revealed, prfA translated

• prfA increases expression of virulence genes

List 4 main ways of regulating translation

• sRNAs

• Thermosensors

• Incorporation of SD into hairpin loop

• Attenuation

What is attenuation?

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

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

Describe the attenuation of the Trp operon

• If trp is scarce, trp-tRNA low

• Trp operon has two trp codons in sequence in short ORF

• If trp low, ribosome pauses, causing formation of 2:3 region hairpin

• Prevents formation of RNAP-pausing hairpin downstream

• Rest of gene is transcribed and translated

• If trp abundant, no pause, 3:4 RNAP-terminating hairpin forms

• Transcription terminated, onward translation reduced as low trp mRNA