BCH311 L11 Ribosome, protein synthesis

similarities between eukaryotic and ribosomal compositions

general function:

• full complex: translation

• small subunit: mRNA binding and codon/anticodon pairing

• large subunit: peptide bond formation

general composition:

• full complex: 2/3 rRNA folded into duplex regions, 1/3 proteins connect rRNAs

• small and large subunit: ONLY rRNA at subunit interface (no protein)

differences between eukaryotic and ribosomal compositions

eukaryotic ribosome composition: full complex 80S

• small subunit: 40S: 30 proteins, 18S rRNA

• large subunit: 60S: 40 proteins, 5S rRNA, 28S rRNA, 5.8S rRNA

prokaryotic ribosome composition: full complex 70S

• small subunit: 30S: 21 proteins (S1-21), 16S rRNA

• large subunit: 50S: 31 proteins (L1-31), 5S rRNA, 23S rRNA

translation initiation: sequence specificity (prok)

translation begins before transcription is complete: co-transcriptional translation

first codon found ~25 nt downstream from 5’ end

ribosome binds ~10 nt upstream at purine-rich Shine Dalgarno (SD) sequence

• recognized by 3’ end of 16S rRNA via bp, helping correctly position initiation codon (AUG) into ribosome’s P site

translation initiation: initiator tRNA + charging (prok)

AUG start codon after the SD signals for incorporation of formyl methionine (fMet) residue

• carried exclusively by the initiator tRNA (tRNA_i^fMet), differing from internal methionines

charging occurs in 2 distinct steps:

1. first, tRNA_i^fMet is charged with unmodified methionine by the regular Met-tRNA synthetase

2. Met-tRNA_i^fMet formyl transferase will attach a formyl group to the N-terminal of the tRNA-bound Met

translation initiation: initiation factors (prok)

IF1, IF2, IF3: P-loop NTPases that undergo significant conformational changes upon binding and hydrolysis of NTPs

translation initiation via IFs occur in 3 key steps

1. IF1 and IF3 binding: IF1 binds near A site of 30S subunit, ensuring tRNA_i binds P site. IF3 prevents premature binding of 50S, preventing formation of 70S complex without mRNA and tRNA_i.

2. binding of mRNA and IF2 to form the 30S initation complex: 16S rRNA in 30S subunit H-bonds mRNA transcript for correct reading frame. IF2-fMet-tRNA_i^fMet-GTP associates with 30S

3. transition to the 70S initiation complex: IF2 bound to 30S subunit undergoes GTP hydrolysis, releasing all IFs from complex. promotes binding of 50S large subunit, resulting in 70S initiation complex

IF2

initiation factor in prokaryotes

in complex with fMet-tRNA_i^fMet and GTP, associates with 30S

when bound to 30S, undergoes GTP hydrolysis, releasing all IFs from complex.

promotes binding of 50S large subunit, resulting in 70S initiation complex

IF1

initiation factor in prokaryotes

binds near A site of 30S subunit, ensuring tRNA_i binds P site

IF3

initiation factor in prokaryotes

prevents premature binding of 50S without mRNA and initiator tRNA

translation elongation stages: general mechanism (prok)

1. aminoacyl tRNA binding: binds at A site, thanks to fMet-tRNA_i^fMet at P site.

2. peptide bond formation: takes place at P site of L subunit. chain is transferred from tRNA in P to tRNA in A. acceptor arms of tRNA undergo positional shift

   • anticodon regions initially remain in the same place

3. translocation: ribosome must shift by 3 nt towards mRNA 3’ end for next codon into A site. driven by GTP hydrolysis and facilitated by elongation factor G (EF-G)

   • after translocation, tRNA anticodons are properly aligned in respective site.

4. cycle: ribosome releases a deacylated tRNA molecule from E site and returns to initial state.

EF-Tu

elongation factor involved in aminoacyl tRNA binding

protects ester linkage between amino acid and tRNA molecule, ensuring aa remains attached to tRNA upon delivery

hydrolyzes GTP to GDP + Pi and releases tRNA molecule to ribosome when correctly paired with mRNA codon

must be regenerated by EF-T

EF-Tu does not interact with initiator tRNA, it is brought to ribosome by IF-2

translation elongation stages: step 1 - aminoacyl tRNA binding (prok)

delivery of amino-acyl tRNA to A site of ribosome facilitated by GTP-bound elongation factor EF-Tu

• EF-Tu protects ester linkage between aa and tRNA molecule, ensuring they are attached

when anticodon correctly pairs with mRNA codon at A site, EF-Tu hydrolyzes GTP to GDP + Pi and releases tRNA to the ribosome

• triggers conformational change that allows tRNA to bind firmly to A site

• EF-Tu is bound to GDP and must be regenerated by guanine exchange factor, EF-Ts

translation elongation stages: step 2 - peptide bond formation (prok)

once A and P site are occupied by aminoacyl-tRNA molecules, peptide bond formation takes place at P site of L subunit

amino group of aa at A attacks carbonyl carbon of aa at P.

• formation of tetrahedral intermediate, polypeptide-chain and aa are linked as well as to tRNAs

• undergoes bond rearrangement, leading to peptide bond formation and deacylated tRNA molecule

upon peptide bond formation, acceptor arms shift positions

• acceptor arm of tRNA with growing pp chain → P site

• deacylated tRNA accept arm → E site

translation elongation stages: step 3 - translocation (prok)

GTP-bound elongation factor EF-G attaches to 50S ribosomal subunit near A site

EF-G hydrolyzes GTP, causing conformational change that simultaneously

• transfers anticodon of tRNA at A site to the P site

• moves the anticodon of the deacylated tRNA at the P site to the E site

ribosome translocates one codon towards 3’ end of mRNA transcript

EF-G

elongation factor involved in translocation

attaches to 50S ribosomal subunit near A site and hydrolyzes GTP, causing translocation-related conformational changes

structure mimics EF-Tu-bound amino-acyl tRNA complex, allowing to fit easily near A site

translation elongation stages: step 4 - cycle

multiple ribosomes can be cycling through the translation elongation process on the same mRNA transcript at the same time

• groupings called polysomes

• enhances translational efficiency for both prokaryotes and eukaryotes

• can be visualized using electron microscopy

mRNA/tRNA pairing: mischarging tRNA molecules

base pairing interactions between mRNA and tRNA codons dictates which amino acid will be added to the growing polypeptide chain

• specific amino acid plays no role in this process

if tRNA is mischarged, anticodon will still interact, and incorrect aa will be incorporated into the polypeptide chain

amino acids do not play a role in codon pairing: experiment

1. tRNA^Cys correctly charged with Cysteine using cysteinyl-tRNA synthetase and ATP

2. the sulfur of the cysteine can then be converted into a H atom using Raney nickel, creating Ala-tRNA^Cys, a mischarged tRNA

3. combining the Ala-tRNA^Cys molecules with a synthetic mRNA template specific for cysteines + the necessary translational machinery produces a polypeptide chain composed of Ala-s

demonstrates that protein synthesis is independent of the amino acid charged to a particular tRNA

wobble hypothesis

codons that differ only in their third base can bind to the same tRNA anticodon

• first two nucleotides of an mRNA codon must form strict WC bp with tRNA anticodon

• 3rd position has greater steric freedom and allows for both Hoogsteen and WC bp between tRNA and mRNA

wobble hypothesis regulation in ribosome

difference in flexibility of 3rd position is dictated by 16S rRNA of the S subunit

features 3 conserved nucleotides that H-bond with position 1 and 2 of the codon, 2 and 3 of the anticodon

• correct H-bonding between these 3 can only occur if WC bp are formed

image: correct WC bp at position 2 enables 16S rRNA to form H-bonds with both anticodon & codon, unlike at position 1. this prevents a conserved nucleotide from making the correct H-bonds, resulting in expulsion. position 3 is not subject to scrutiny

translation termination (prok)

no aminoacyl-tRNA molecules with anticodons complementary to 3 STOP codons; UAA, UGA, UAG.

when encountered at A site of the ribosome, they are instead recognized by release factors (RFs)

1. binding of release factor: when STOP codon is encountered in A site, RF-1 or RF-2 binds assisted by GTP-bound RF-3. RF will carry a molecule of water with them

2. peptide release: water molecule provided by RF used by ribosome to hydrolyze ester bond between pp chain and P site tRNA, releasing polypeptide

3. complex dissociation: RF-3 hydrolyzes GTP to GDP, leading to dissociation of RF. ribosomal release factor (RRF) binds ribosome and triggers release of entire translation complex by GTP hydrolysis

RF-1, RF-2, RF-3, RRF

RF-1: recognizes STOP codons UAA and UAG

RF-2: recognizes STOP codons UAA and UGA

both carry a water molecule into the A site to hydrolyze the ester bond between pp chain and tRNA in P site

RF-3: hydrolyzes GTP→GDP, leading to dissociation of RFs

RRF: hydrolyzes GTP→GDP, binds to the ribosome and triggers the release of the entire translation complex

prokaryotic translation ATPs GTPs for 9 amino acid pp chain

1. charging tRNAs: 2 molecules per charging event - 2×9=19 ATP

2. translation initiation: 1 molecule to bring in initiator tRNA - 1 GTP

3. aminoacyl-tRNA binding: 1 molecule for additional amino acids brought in by EF-Tu, hydrolyzed upon binding - 1×8=8 GTP

4. translocation: EF-G and GTP hydrolysis for each translocation - 1×8=8 GTP

5. translation termination: requires 1 GTP from RF-3 - 1 GTP

6. ribosome disassembly: occurs using RRF and GTP hydrolysis - 1 GTP

18 ATPs and 19 GTPs required to synthesize a 9 amino acid protein

translation initiation: step 1 - assembly of complexes (euk)

independent assembly of two essential complexes:

eIF4F heterotrimer complex

• includes eIF4E (binds to 7-methyl-G cap of mRNA)

43S pre-initiation complex

• consists of small ribosomal subunit, Met-tRNA_i^Met, and several eukaryotic initiation factors (eIFs, like eIF2)

  • eIF2, bound to GTP, facilitates interaction between tRNA_i and S subunit

  • tRNA_i^Met differs from tRNA species that carries internal methionines, but is not formylated. associates with S subunit devoid of mRNA

translation initiation: step 2 - complexes bind mRNA (euk)

both complexes bind to the mRNA at the 5’ cap

• eIF4F, which binds to 7-me-G cap of mRNA

• 43S pre-initiation complex, which consists of several initiation factors and tRNA_i^Met

eIF4F complex attaches first through eIF4E subunit

followed by binding of 43S pre-initiation complex

translation initiation: step 3 - circularization of mRNA (euk)

once both complexes are bound to the mRNA, transcript undergoes circularization

thought to improve ribosomal recycling by facilitating the re-binding of ribosome after the mRNA is finished translating

eIF4G component of eIF4F complex acts as bridge between cap-binding protein eIF4E and poly(A)-binding protein at 3’ end of mRNA

translation initiation: step 4 - scanning of mRNA (euk)

circularization initiates 5’ to 3’ scanning of the mRNA by the S subunit in search of the start codon

scanning driven by helicases and ATP hydrolysis

start codon is simply the first AUG located by the complex, closest to the 5’ end

translation initiation: step 5 - recognition of initiation codon (euk)

when first AUG is encountered by S subunit, interaction between mRNA codon and cognate tRNA anticodon triggers GTP hydrolysis of eIF2

hydrolysis facilitates binding of L (60S) subunit and positioning of tRNA_i at P site, forming 80S ribosome

once assembled, all eIFs dissociate, ribosome is now ready to proceed to the elongation phase

puromycin: ribosomes as drug targets

closely resembles A of tRNA acceptor arm (CCA), attached to an amino acid. features amide linkage instead of ester linkage

allows puromycin to fit into A site of ribosome as though it were a charged tRNA to be incorporated into pp chain

its N atom attacks C-terminal end of growing pp chain - forming normal peptide bond - pp chain is transferred to puromycin

incoming aa is unable to attack during the next round of translation elongation due to amide linkage

puromycin binds weakly to ribosome, leading to dissociation and halting of translation

affects both eukaryotes and prokaryotes

streptomycin: ribosomes as drug targets

streptomycin prevents correct binding of tRNA_i in prokaryotes

due to streptomycin’s basic nature, conferred by many amino groups

diptheria toxin: ribosomes as drug targets

affects eukaryotes

upon entering cell, toxin is cleaved into two fragments: A and B

• A fragment acts as enzyme that catalyzes NAD+ cleavage into ADP-ribose and nicotinamide

toxin attaches ADP-ribose moiety to modified histidine residue in elongation factor 2 called dipthamide

• modification inhibits EF2 ability to translocate to ribosome during protein synthesis, halting transformation

ricin: ribosomes as drug targets

N-glycosidase that inhibits protein synthesis in eukaryotes

heterodimer composed of two chains

• A chain, has catalytic activity

• B chain, responsible for cellular uptake, connected to A chain by single disulfide bond

when taken up by cells through endocytosis, A chain is released into cytoplasm

• specifically targets a conserved adenine residue in 28S rRNA of ribosome, cleaving N-glycosidic bond between base and respective ribose sugar

• prevents binding of EFs and inactivates ribosome