Basics of Translation

Prokaryotic Biology Unit 3

What is translation?

Synthesis of polypeptide from genetic information in mRNA

What structures are involved in translation? (general)

• Ribosomes (comprised of rRNAs and proteins)

• Amino Acids

• mRNA and tRNA

• Initiation, Elongation, and Termination factors

What phases are involved in translation and what occurs at each phase?

Phases include initiation, elongation, termination, and ribosome recycling. Regulation occurs at each phase

What is the ribosome?

A ribonucleoprotein complex that synthesizes the protein (translates mRNA). It binds to the mRNA, tRNA, and directs peptide bond formation. It contains ~56 proteins and 3 RNAs (rRNAs). Many of the ribosomal proteins are non-essential, although loss of these proteins often results in slow growth and/or cold-sensitivity.

What are the three tRNA binding sites?

• A Binding Site (aminoacyl-tRNA)

• P Binding Site (peptidyl-tRNA)

• E Site (Exit Site)

What are the three ribosomal complex?

• 30S (small)

• 50S (large)

• 70S (30S plus 50S)

What is the small ribosomal subunit?

• 30S

• Protein: 22 protein subunits labeled S1-S22

• RNA: 16S RNA, critical for initiation (1540 nt)

What is the large ribosomal subunit?

• 50S subunit

• Protein: 34 proteins (L1-L36)

• RNA: 5S (120 nt) and 23S (2900 nt)

• Contains a catalytic site where the peptide bonds are formed

What is the A site within the ribosome?

incoming tRNA site (acceptor)

What is the P site within the ribosome?

contains tRNA to which the nascent polypeptide is attached (peptidyl)

What is the E site within the ribosome?

exit site where the empty tRNA is discharged

What does the 23S rRNA do?

catalysis (peptide bond formation) making this a ribozyme

How do antibiotics target the ribosome?

• Ribosome inhibitors constitute over half of the antibiotics currently used to treat bacterial infections.

• Such antibiotics bind the A, P, E sites or nascent peptide exit channel (NPET)

• Can inhibit binding of tRNAs, formation of 30S inititation complex, peptide bond formation, or function of translation factors EF-Tu, EF-G and IF2

What are the mechanisms of resistance to ribosome inhibitors?

• Resistance to naturally produced antibiotics is inherently linked to their production

• Pathogens frequently acquire genes on mobile elements (plasmids, transposons, integrons) that confer antibiotic resistance through horizontal gene transfer

  • Use efflux pumps to keep the intracellular concentration of the drug low (ex. tetracycline-specific efflux pumps which aren’t effective against 3rd generation tetracyclines)

  • Degrade or enzymatically modify the drug (ex. enzymatic acetylation of chloramphenicol reduces its toxicity)

  • Alteration of the target site by chemical modification or mutation (ex. methylation of adenine 2058 in the 23S rRNA by ErmE confers resistance to erythromycin)

  • Ribosome protection proteins (TetO and TetM) are paralogues of EF-G that bind to the tetracycline-stalled ribosome and cause tetracycline to dissociate from the ribosome

What is the Shine-Dalgarno sequence?

• an AG-rich region ~8 nt upstream of start codon for gene (only in bacterial and many archaeal mRNAs whereas eukaryotes utilize Kozak sequence)

• Seldom have a “canonical” SD sequence (there is degeneracy in the SD sequences of mRNAs)

• This is not the ribosome binding sites, just a part of the site as the RBS spans 20 to +15, while the SD is only ~7nt in length

• Each ORF in mRNA has its own RBS - translation terminates at preceding ORF and re-initiates at next ORF

• Some mRNAs lack a 5’-UTR - follow pathways for translation initation that differ from the pathway for SD-leader mRNAs

What is the start codon found on mRNA?

Start codon is usually AUG, but can be GUG or UUG

Regardless of which codon is used, N-formylmethionine (fMet) is the amino acid incorporated at that position. The tRNA for start codon (tRNA^fMet) differs from tRNA for protein elongation (tRNA^Met).

fMet is often removed from the polypeptide chaine by a methionine aminopeptidase

What features of mRNA determine translational efficiency?

• Codon used for initiation (AUG, GUG, or UUG)

• SD Sequence

• Spacer between SD Sequences and Start Codon

• mRNA secondary structure near the start site - small subunit protein bS1 (b → bacteria, S → small 30S) that acts as an RNA chaperone to unfold structured mRNA on the ribosome

Complete the sentence: Individual elements of a RBS have limited, context-dependent effects on translation initation, meaning their

impact on efficiency is heavily influenced by surrounding mRNA structural features, such as secondary structures

What are tRNAs?

Transfer RNAs, which are small RNAs that bring amino acids to the ribosome and decipher the code for synthesizing proteins contained in the mRNA

It has an anticodon that base pairs with the codon in the mRNA

Some codons are used more frequently that others (6 codons for arginine; in E. coli, two of these are used for 72% of Arg codons)

Many of the nucleosides within tRNA are post-transcriptionally modified (Psi and D) by a set of tRNA modifying enzymes - contribute to the functional and structural integrity of the RNA molecules.

What is the wobble position?

Multiple codons for a single amino acid tend to differ in the 3rd position on mRNA (1st position in tRNA)

How do the first two nucleotides in a codon interact with the anticodon?

Through Watson-Crick base pairing rules (A-U and C-G)

How does the thrid nucleotide in a codon interact with the anticodon?

Through wobble rules

Anticodon nucleotide that base pairs with the third nucleotide in codon often modified to accommodate wobble base pairing (ex. uridine-5-ocyacetic)

Allows faster dissociation of tRNA from mRNA

Does not apply to the initiation codon (AUG, UUG, GUG) → it’s the first nt

What does the free 30S have bound to it in order to prevent 50S binding during translation intiation?

IF3 protein

What only binds N-formymethionine tRNA^fMet?

IF2•GTP

formyl group of fMet is a positive determinant for IF2 binding (promotes initiation) and a negative determinant for Ef-Tu binding (prevents elongation)

What is able to bind in the P site of the 30S preinitation complex?

IF2•GTP-fMet-tRNA^fMet ternary complex

What two initation factors cooperate to ensure that only the initator tRNA binds to the P site?

IF1 cooperates with IF2

also occludes aminoacyl-tRNAs from A site until 70s initation complex has formed

What binds to the mRNA during translation initation?

30S, IF2•GTP-fMet:tRNA^fMet , and IF1

What allows 50S subunit to bind to the 30S subunit?

IF3 is released which allows 50S subunit to bind

What occurs after 50S subunit clamps down?

50S subunit clamps down, hydrolysis of GTP by IF2•GTP, release of IF1 and IF2 from ribosome

When can mRNA enter the 30S preinitiation complex?

Any step in the initation pathway

it is independent of presence of initation factors or fMet-tRNA^fMet

Why do you think the spacing between the SD sequence and start codon is important for efficient translation?

the spacing ensures that the start codon sits in the P site during initation

Why is resistance to naturally produced antibiotics inherently linked to their production?

It allows the inhibition of other antibiotics around it, but it must prevent growth inhibition of itself

What does EF-Tu bind?

EF-Tu binds GTP and charged tRNA, but does not bind tRNA^fMet

What is a charged tRNA?

tRNA with amino acid covalently attached

EF-Tu•aa-tRNA^AA is delivered to the A site in the 50S subunit. If the codon-antidon interactions are correct, what happens next?

hint: referred to the proofreading step

GTP bound to EF-Tu is hydrolyzed to GDP and EF-Tu leaves the ribosome

Fidelity of aa-tRNA selection is _______

high, but not error-proof (error frequency <10^-3)

After Ef-Tu GTP is hydrolyzed, what happens next?

Nascent polypeptide on tRNA in P site is transferred to tRNA in A site

• Involes 23S rRNA (not protein catalyzed)

What does EF-G•GTP do?

It interacts with ribosome, hydrolyzes GTP, facilitates physical movement of ribosome down one codon

• tRNA in A site moves to P site

• Uncharged tRNA in P site moves to E site from which it exits the ribosome

What is the first step of EF-Tu recycling?

After tRNA^AA delivery, EF-Tu is GDP bound

What is the second step of EF-Tu recycling?

EF-Ts required to remove GDP from EF-Tu

What is the third step of EF-Tu recycling?

Fresh GTP can bind EF-Tu and displace EF-Ts

What is the fourth step of EF-Tu recycling?

Newly-GTP-bound EF-Tu can now bind tRNA^AA for 70S delivery

What is the peptidyltransferase reaction?

Nucleophilic attack by amine group of amino acid on tRNA in the A-site on carboxyl group of amino acid of peptide attached to tRNA in P-site

Occurs in the active site fromed by 23S rRNA, not the ribosomal proteins

the entire peptide transferred to tRNA in the A site

Where do ribosomes commonly stall in polypeptide synthesis?

poly-proline stretches (PP-motifs) → multiple prolines in a line

What occurs once a ribosome interacts with a poly-proline stretch?

Binding of peptidyl-tRNA in P site is destabilized, may lead to the release of peptidyl-tRNA

How is the stalling relieved during poly-proline stretches?

Stalling is relieved by EF-P, which binds to E site and interacts with P site tRNA^Pro to stabilize tRNA and nascent polypeptide chain by forcing prolines to adopt a conformation that can pass into the exit channel

What is the benefit to ribosomal pausing that is caused by the PP-motifs?

Slows down translation to allow more time for protein to fold correctly

When does a ribosome stop incorporating amino acids?

When the ribosome encounters a STOP codon in the A site - UAA; UAG; UGA

What recognizes stop codons in the A site?

Hint: no tRNAs for stop codons

Protein release factors RF1 or RF2

Their binding stimulates release of polypeptide from tRNA in P-site

What stop codons does the RF1 protein recognize?

UAA, UAG

What stop codons does the RF2 protein recognize?

UAA, UGA

What does RF3 do?

Stimulates release of RF1/RF2 from the ribosome

Paper detailed role in post peptidyl quality control

What is required for dissociation of 70S ribosome?

Ribosome recycling factor and EF-GꞏGTP enters A site, GTP hydrolysis by EF-G is required for dissociation of 70S ribosome

What re-engages with with the 30S subunit after the 70S ribosome is dissociated?

IF3 re-engages 30S subunit to remove tRNA and mRNA

What charges tRNAs?

aminoacyl-tRNA synthetases (aaRS) in a two-step reaction

What does the aaRS do?

Recognizes a specific amino acid (usually) AND anti-codon on the cognate tRNA and covalently attaches the amino acid to the 3’-end of tRNA

What is unique about E. coli’s aaRS

It has a unique aaRS for each amino acid, with the exception of lysine

• 2 different aaRSs for lysine (LysRS and LysU)

MetRS is used to charge…

both tRNA^Met and tRNA^fMet - tRNA^fMet is first charged with methionine then formylated

aaRSs in eukaryotes

Most eukaryotic cells have two separate sets of aaRSs (one in cytoplasm and one in mitochondria), while plants have a third set in the chloroplasts

What is the editing domain in aaRSs?

About half of the aaRSs in E. coli have this that removes the amino acid from tRNAs that have been charged with the wrong amino acid - aaRSs that are prone to mistakes due to the cognate amino acid being easily confused with similar, non-cognate amino acids

What occurs during the “backreaction” of aaRSs?

Some aaRSs synthesize adenyl dinucleotides (alarmones) during the backreaction of the first step in charging the tRNA in which pyrophosphate (PPi) is replaced with ATP (or less often ADP, GTP, and GDP)

In bacteria, AppppA is thought to modulate the…

heat-shock response - binds to various heat-shock proteins and inhibits the activity of at least one of these proteins (DnaK)

What is the difference between LysRS and LysU in E. coli?

LysRS is constitutively expressed

LysU is heat-inducible

Both catalyze this backreaction. LysU is more thermostable than LysRS and is throught to help to maintain a pool of adenyl dinucleotides during prolonged heat shock.

What products would be formed with ATP, ADP, GTP, and GDP during the “backreaction”?

ATP: APPPPA

ADP: APPPA

GTP: APPPPA

GDP: APPPA