Translation: Genetic Code and Protein Synthesis PDF

The central dogma  Polypeptide chains have a polarity By convention sequence of amino acids is given from the amino to the carboxyl terminal end from left to right. Different levels of protein structure  Peptide bonds between adjacent amino acids Between...

The central dogma  Polypeptide chains have a polarity By convention sequence of amino acids is given from the amino to the carboxyl terminal end from left to right. Different levels of protein structure  Peptide bonds between adjacent amino acids Between every 4th amino acid Between amino acids further away in parallel strands Hydrogen bonds between atoms of the polypeptide backbone Between amino acids in the same polypeptide chain Between amino acids from different polypeptide chains Different types of bonds between the side chains of amino acids Hydrogen bonds Ionic bonds Hydrophobic interactions Disulfide (covalent bonds) between –SH groups of cysteines  1960s- Nirenberg, Leder, Mathaei, and Khorana deciphered the genetic code How many nucleotides are read as an amino acid? There are 20 different amino acids in proteins 41= 4 different amino acids 42= 16 different amino acids 3 nucleotides make up a codon 43= 64 different amino acids  Is there overlapping of codons?  Is there punctuation in the code? Poly U codes for UUU codes for phenyalanine polyphenylalanine  Genetic code Made up of trinucleotides, codons 43=64 possible codons only 20 amino acids 3 stop codons, 61 codons 20 amino acids Redundant or degenerate More than 1 codon code for the same amino acid Non-overlapping Non-punctuated UNIVERSAL GUC codes for valine  Stop 5’ 3’ If we don’t know which strand is the coding strand in the DNA Which one is the correct reading frame?  Which is the correct reading frame? DNA sequence of the nonmutant segment of an E. coli gene: Amino acid sequences of the protein encoded by the gene from nonmutant and 2 mutant strains: Gly Arg GGN CGN AGA AGG Glu GAA GAG Note: This example is very similar to the one you have in Genetic Analysis 9.3 The following segment of DNA encodes a peptide. 5′-... CCCAGCCTAGCCTTTGCAAGAGGCCATATCGAC...-3′ 3′-... GGGTCGGATCGGAAACGTTCTCCGGTATAGCTG...-5′ carboxy Gly Lys Cys Ser Ala Met amino How do we know what is the sequence of the peptide? Which is the coding strand? Which is the reading frame? We look for a start codon (ATG) and after that there should be an Open Reading Frame (ORF)- series of codons coding for amino acids, without codonaATG stop in codon in the same frame. No start the top strand. There is an ATG in the bottom strand, so the bottom strand is the coding strand. Reading in the same frame as the start codon there are 5 additional codons coding for amino acids followed by a stop codon so translating those codons will give us the peptide sequence. Note: Genetic analysis 9.2 in your book  In eukaryotic cells In prokaryotic cells Operon-Group of genes ▪ encoding proteins with related functions ▪ sharing one promoter tRNAs tRNA + amino acid= amino acyl tRNA  Amino acyl tRNA synthetase is the enzyme that attaches an amino acid to a tRNA The enzyme is very specific, so it will only attach a specific amino acid to a given tRNA a tRNA with certain anticodon will only get a specific amino acid attached by the enzyme to its 3’ Watch the video to see how the enzyme works: https://www.youtube.com/watch?v=fcltHCAEwK0  A codon in the mRNA interacts with an anticodon in the tRNA. The two polynucleotide chains will be antiparallel, so the 3’ base of the codon interacts with the 5’ base of the anticodon. The interaction between the 3’ base of a codon in the mRNA and the 5’ base of the anticodon is more flexible than those in the B DNA: Wobble pairing. One tRNA with a given anticodon can interact with several different codons. tRNA-Ser1 can interact with UCU and UCC both of these codons code for serine tRNA-Ser2 can interact with UCA and UCG both of these codons code for serine tRNA-Ile can interact with AUC, AUA and AUU, so all these 3 codons code for 3’ 5’ 3’ 5’ 3’ 5’isoleucine 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Each of the codons coding for aspartic acid interact with a different anticodon. © 2015 Pearson Education, Inc.  Some tRNAs interact with several codons redundancy of the genetic code Some tRNAs interact with only one codon There are less than 61, but more than 20 different tRNAs in the cell.  Ribosomes S- Svedberg units- a measure of a particle's size based on its sedimentation rate Three sites to which tRNAs can bind in the ribosomes: A site- Site where an Amino acyl tRNA binds initially to the ribosome and interacts with the corresponding codon in the mRNA (except for the Met-tRNA binding to the start codon). P site- Site where a Amino acyl tRNA: tRNA with an Peptidyl-tRNA is when amino acid attached to the 3’ the new amino acyl tRNA end- also called charged tRNA. binds to the A site. Peptidyl-tRNA: tRNA with a E site- Exit site, from peptide attached to its 3’ end. which an empty tRNA leaves the ribosome. Empty tRNA: tRNA without an amino acid or peptide bound.  Translation: Initiation, Elongation and Termination Initiation The mRNA binds to the small ribosomal subunit and the start codon is positioned in the P site. ▪ In prokaryotes, a conserved sequence in the 5’-UTR of the mRNA, the Shine-Dalgarno sequence, binds to a rRNA in the small ribosomal subunit. ▪ In eukaryotes, the cap at the 5’ end of the mRNA makes it bind to the small ribosomal subunit. The initiator tRNA binds to the start codon in the P site. ▪ In prokaryotes the initiator tRNA that binds to the start codon carries a formyl-Met ▪ In eukaryotes the initiator tRNA carries a Met The large ribosomal subunit assembles with the small subunit. Energy is required, provided by hydrolysis of GTP. Initiation factors (different in prokaryotic and eukaryotic cells) are required. Initiation in bacteria Initiation in eukaryotes Please follow the link to watch an initiation of translation animation https://mediaplayer.pearsoncmg.com/assets/secs-initiation-of-translation Elongation A new amino acyl-tRNA binds to the next codon, so the amino acyl-tRNA is positioned in the A site. The amino acid or peptide on the tRNA on the P-site is transferred to the charged tRNA on the A site, and a peptide bond is formed between the amino acid attached to the tRNA on the P site and the one attached to the tRNA on the A site A peptidyl tRNA is now on the A site, and an empty tRNA is on the P site. There is translocation or sliding of the ribosome on the mRNA toward its 3’ end. ▪ The peptidyl-tRNA on the A site moves to the P site. ▪ The empty tRNA on the P site moves to the E site. The elongation cycle is repeated as many times as amino acids are added. Energy is required- obtained from hydrolysis of GTP. Figure 11.25 Elongation in bacteria Please follow the link to watch a translation elongation animation. https://mediaplayer.pearsoncmg.com/assets/secs-elongation-of-the- polypeptide-chain Termination When a stop codon is on the A site, no tRNA can bind to the stop codon so translation stops. The polypeptide bound to the tRNA on the P site is released. The mRNA is released from the ribosome. The ribosomal subunits separate. Energy is required-obtained from hydrolysis of GTP. Termination/release factors are required, which are different between eukaryotic and prokaryotic cells. Figure 11.29  In translation generally there are multiple ribosomes attached to the same mRNA molecule, forming polyribosomes or polysomes. In bacteria transcription and translation of the same mRNA are happening simultaneously, so polyribosomes are seen on mRNA before its translation is completed