Translation - Douma

Open Reading Frame ORF

Begins with start codon (AUG), ends with at stop codon (UAU UAG UGA)

tRNA Structure

Acceptor Stem is the region of tRNA that binds to the corresponding amino acid, ensuring accurate translation during protein synthesis. Anticodon Loop contains the anticodon which binds to the mRNA codon sequence.

Wobble Base

Wobble Base refers to the flexible base pairing at the third position of the codon-anticodon interaction, allowing a single tRNA to recognize multiple codons.

Wobble Base Special Pairing

Two codons recognized: G recognizes C and U, U recognizes G and A.

Three codons recognized: I (post-transcriptionally added) recognizes A U C.

Aminoacyl-tRNA synthetases

Add amino acid to 3’ end of tRNA creating charged tRNA. This synthetase binds tRNA, then transfers the correct AA to the 3’ end of tRNA using codon / anticodon pairing through the utilization of ATP hydrolysis for energy, forming a covalent bond and charged tRNA.

Prokaryotic Initiation Factor Sequence

IFs bind to small ribosomal subunit, block large ribosomal subunit from binding and blocks charged tRNA from coming in. mRNA base pairs with rRNA at Shine Delgarno sequence to position 5’ AUG at P-Site, then IF brings charged tRNA carrying N-formylmethionine (fMet) into P-Site by using GTP.

Shine-Dalgarno Sequence

mRNA sequence where ribosomal rRNA binds, in 5’ UTR / upstream of AUG, allows AUG to bind to anticodon of tRNA carrying fMet.

Translation Initiation Complex

A Site Vacant, P Site fMet, E site vacant, mRNA codon AUG in P-site. Once GTP is hydrolyzed from IFs IFs come off, large ribosomal subunit binds to small ribosomal subunit, complex ready for elongation.

Prokaryotic Elongation Factors

Involved in making peptide bonds. EF-Tu delivers AAs to A-site. EF-Tu binds GTP and tRNA, when GTP is hydrolyzed to GDP it releases tRNA into the A-Site. EF-Ts recycles EF-Tu-GDP to EF-Tu-GTP, as EF-Ts is a nucleotide exchange factor.

Peptide Bonds and Translocation

Ribosome catalyzes formation of peptide bonds using rRNA activity, alpha amino group of A site attacks carboxylic end of fMet in P-Site to form bond, while this is done the ester bond between fMet and 3’ tRNA is broken, dipeptide exists on A-site tRNA.

EF-G promotes translocation, binds GTP near A-Site, when hydrolyzes GTP it pushes each site down 1 spot, Dipeptide in P-Site, A-Site open, this opens up new codon site.

Translation Termination

Occurs when reaches UAU, UAG, UGA stop codon. Stalls ribosome bc no tRNA matching, termination factors release, bind to A-site and trick ribosome, carboxylic end of P-site peptide reacts with water, ester bond breaks and peptide is released, ending translation.

Ribosome Recycling Factors RRF bind to ribosome complex, make it fall apart back into its individual pieces to promote translation again.

Eukaryotic Difference in Translation (Initiation)

eIFs, eIF4F complex binds mRNA 5’ cap, also binds to polyA tail via polyA binding protein (PABP), circularizing mRNA. Other eIFs same as IFs. If circularization occurs translation will begin as the RNA was processed properly.

Eukaryotic Initiation

Bring in tRNA first, before mRNA comes in bring in tRNA attached to Met (not fMet), place tRNA in P-Site. eIF4F connects mRNA to rRNA after tRNA is in P-Site. Ribosome scans mRNA 5’-3’ to find AUG with Kozak sequence. tRNA with anticodon in P-Site, know mRNA in right spot when AUG binds. Tells eIFs translation is ready, hydrolyzes GTP, falls off and all processes begin.

Antibiotics Target rRNA (Structure)

Antibiotics have selective toxicity, target specific 2ndary structures in prokaryotic rRNA that differs from our rRNA. 4

4 Antibiotics affecting Translation

Chloramphenicol - Blocks peptidyl transferase activity.

Tetracycline - Blocks A-Site entry of tRNA.

Erythromycin - Prevents translocation.

Streptomycin - causes mRNA to be misread.

Chloramphenicol Toxicity

Typhoid fever treated with chloramphenicol which inhibits peptidyl transferase. Prolonged use can also affect mitochondrial translation / ribosomes in human cells, leading to impaired protein synthesis / blood marrow suppression.