Translation - Introduction (BMS100_BCH1-07_F22)

Translation - introduction • Following processing and termination of transcription, mature mRNA is exported from the nucleus through nuclear pore complexes. • Once in the cytosol, mature mRNA is translated into protein Translation – introduction cont. • The mRNA sequence is decoded in sets of 3 nu...

Translation - introduction • Following processing and termination of transcription, mature mRNA is exported from the nucleus through nuclear pore complexes. • Once in the cytosol, mature mRNA is translated into protein Translation – introduction cont. • The mRNA sequence is decoded in sets of 3 nucleotides called codons. § There are 64 possible combinations of of 3 nucleotides but only 20 amino acids • Some amino acids are specified by more than one codon, demonstrating the redundancy of the genetic code Translation – Reading Frame • Reading Frames: § Since mRNA is interpreted in 3-nucleotide codons, the correct polypeptide sequence depends on the correct reading frame. • Special punctuation is needed to determine the correct reading frame. § Preview – in Eukaryotes it is the first AUG sequence. In Prokaryotes it is the Shine Dalgarno sequence Thinking Question: • Reading Frames: § Here are three possible polypeptides based on different reading frames Q: What might happen if a nucleotide was accidentally inserted into the middle of a gene? Translation – What is needed • A number of molecules are needed for translation: § mRNA transcript § tRNA • Needs to be bound to the correct amino acids (“charged tRNA”) § Ribosomes • A large and small subunit Translation – Preparing the tRNA • The cell makes a variety of tRNAs, each corresponding to one of the 20 amino acids § The enzyme aminoacyl-tRNA synthetase catalyzes the attachment of correct amino acid to tRNA Translation – What is needed • Protein synthesis is performed in the ribosome § Helps maintain correct reading frame and ensure accuracy of codon – anti-codon interaction • The ribosome complex is composed of various ribosomal proteins and ribosomal RNA (rRNA) § 2 subunits: • Small subunit • Large subunit *Note the 4 binding sites on the ribosome Translation – What is needed • Translation can be divided into 3 steps: § 1. Initiation § 2. Elongation • • • • A) tRNA binding B) Peptide bond formation C) Large subunit translocation D) Small subunit translocation § 3. Termination Translation – Steps • Translation can be divided into 3 steps: § 1. Initiation § 2. Elongation • • • • A) tRNA binding B) Peptide bond formation C) Large subunit translocation D) Small subunit translocation § 3. Termination Step 1 - Initiation • AUG is the first codon translated on the mRNA § Initiator tRNA carries the amino acid methionine • *Thus, all newly made proteins begin with methionine as the first amino acid at their N-terminus § Forms an initiator tRNA-methionine complex (Met-tRNAi) • Met-tRNAi is loaded into the small ribosomal subunit with initiation factors (eIFs) Step 1 – Initiation cont. • Small ribosome binds to the 5’ end of the mRNA § The 5’ 7-methyl guanosine cap helps with recognition of the 5’ end Step 1 – Initiation: scanning mechanism • Small ribosome moves along the mRNA (from 5’ to 3’) scanning for the first AUG § Requires ATP hydrolysis • Initiation factors dissociate & the large ribosome subunit assembles to complete the ribosome complex Step 1 – Initiation: Prokaryotes • Prokaryotic mRNA is polycistronic § An additional recognition sequence is needed for ribosome binding • Shine-Dalgnaro sequence (aka ribosome-binding site) Step 2 – Elongation • A) tRNA binding § Newly charged tRNA binds to the A site of the ribosome complex • B) Peptide bond formation § Carboxyl end of the polypeptide chain is released from the tRNA at the P site & joins the amino acid linked to the tRNA at the A site § This new peptide bond is catalyzed by peptidyl transferase enzyme contained within the large ribosomal subunit. Step 2 – Elongation • C) Translocation of large subunit: § Large ribosomal subunit moves relative to the mRNA held by the small subunit § Two tRNAs are shifted to the E and P sites • D) Translocation of small subunit § Small subunit shifts by 3 nucleotides § tRNA in E site is ejected • Cycle is repeated for new incoming amino acyl-tRNA Step 2 – Elongation • Elongation proceeds efficiently and accurately with the help of elongation factors (EFs) § These elongation factors enter and leave the ribosome during each cycle & are coupled with GTP hydrolysis Step 3 – Termination • A STOP codon marks the end of translation § UAA, UAG, UGA • Not recognized by a tRNA & do not specify an amino acid • Release factors bind to ribosomes with a stop codon in the A site Step 3 – Termination • Peptidyl transferase catalyzes the addition of a water molecular rather than amino acid § This frees the carboxyl end and releases the polypeptide • Ribosome releases the mRNA & dissociates into the large and small subunits § Subunits are recycled to begin new round of protein synthesis Knowledge Check • The first amino acid added to any polypeptide is: § A) Leucine § B) Methionine § C) Phenylalanine § D) Lysine Knowledge Check • Which of the following describes the correct order of translation initiation in eukaryotes: § A) Small ribosomal subunit binds to the 5’ cap and scans the mRNA until it reaches AUG, then Met-tRNAi binds to the P site § B) Met-tRNAi binds to the P site of the small ribosomal subunit, small ribosomal subunit binds to the 5’ cap and then scans mRNA until it reaches AUG § C) Small ribosomal subunit binds to the 5’ cap & scans the mRNA until it reaches AUG, the large ribosomal subunit binds, and then Met-tRNAi binds to the P site. § D) Small ribosomal subunit binds to the Shine Dalgnaro sequence, Met-tRNAi bind to the small ribosomal subunit, an then the small ribosomal subunit scans the mRNA until it reaches AUG Polysomes • Synthesis of proteins occurs on polyribosomes (or polysomes) § Multiple initiations take place on each mRNA molecule being translation § As soon as the preceding ribosome has translated enough of the nucleotide sequence to move out of the way, a new ribosome complex is formed. § Helps speed up rate of protein synthesis Post-translational • Once full translated, what happens next? § Protein is folded into specific 3-D shape • Proper folding is important since structure of a protein dictates its function! § More to come next class § May be modified in the ER: • Eg. Glycosylated – addition of mono- or oligosaccharide § Sent to its proper cellular location Clinical Application • Many antibiotics function by inhibiting bacterial protein synthesis § Interfering with the correct functioning of prokaryotic ribosomes § For example: