Protein Synthesis PDF

PROTEIN SYNTHESIS EXPRESSION OF THE GENOME PROFESSOR W. MCLAUGHLIN  Translation  Genetic information contained within the order of nucleotides in the mRNA generate the linear sequences of amino acids in proteins  It is the most highly conserved across all organisms ...

PROTEIN SYNTHESIS EXPRESSION OF THE GENOME PROFESSOR W. MCLAUGHLIN  Translation  Genetic information contained within the order of nucleotides in the mRNA generate the linear sequences of amino acids in proteins  It is the most highly conserved across all organisms  The most energetic process  Translation Machinery  mRNA  Organization of nt sequence  tRNA  Structure of tRNA  Aminoacyl tRNA synthetase  Recognition and attachment of correct aa to each tRNA  Ribosome  Decode the nt seq and the addition of aa to the polypeptide chain  mRNA  The protein coding region of each is composed of a contiguous, non-overlapping string of codons called an open reading frame (ORF)  Each ORF specifies a single protein  Translation starts at the 5’-end and proceeds one codon at a time to the 3’-end  The first and last codons – start and stop codons  ORF – Start Codon  In bacteria the start codon is usually 5’-AUG-3’  5’-GUG-3’ and 5’-UUG-3’ are also used  Eukaryotic cells always use 5’-AUG-3’  The start codon is the first aa to be incorporated into the polypeptide chain  Defines the reading frames for all subsequent codon  Stop Codons  Defines the end of the open reading frame and signal termination of polypeptide synthesis  The stop codons are 5’-UAG-3’, 5’-UGA-’3 and 5’- UAA-3’ Reading frames  mRNA  Prokaryotic mRNA usually contain two or more ORFs – polycistronic mRNA  Encodes proteins with related functions  Operon  Eukaryotic mRNA always contain a single ORF – monocistronic mRNA  Prokaryotic mRNA  Many prokaryotic ORFs contain a short sequence 3 to 9 bp upstream of the start codon - RBS  This region is also called the Shine-Dalgarno sequence  The RBS sequence is complementary to sequence located near the 3’-end of the 16S rRNA  RBS  The RBS has the sequence 5’–AGGAGG-3’  The 16S rRNA has the sequence 5’-CCUCCU-3’  The RBS base pairs with the 16S rRNA at this sequence and align the ribosome with the beginning of the ORF  The extent of complementarity and the spacing between the RBS and start codon with influence how actively an ORF is translated  Eukaryotic mRNA  Recruits the ribosome using the 5’-cap  5’-cap is a methylated guanine nt that is joined at the 5’-end of the mRNA through 3 phosphate groups – unusual 5’ – 5’ linkage  Once the ribosome attaches it moves 5’ to 3’ until it encounters a 5’-AUG-3’ through a process called scanning.  The efficiency of translation in eukaryotic mammalian mRNA is increased by   the presence of a purine 3 bases upstream of the start codon and a guanine immediately down stream – 5’-G/ANNAUGG-3’ – Kozac sequence  Also by the presence of the polyA tail at the 3’-end  Transfer RNA  Adapters between codons and amino acids they specify  There are many types of tRNA each carrying a specific amino acid and each recognizing a specific codon or codons in the mRNA  Features of the tRNA  75 to 95 nt in length  All end at the 3’-terminus with 5’-CCA-3’  attached to the aa by the enzyme aminoacyl tRNA synthase  Not encoded by the tRNA gene but added by the CCA adding enzyme  Contain several unusual bases  Psuedouridine, dihydrouridine  All have a common secondary clover structure  tRNA  Acceptor arm  site of attachment of aa  Three stem loops  D loop, U loop, anticodon loop  Variable loop  Size between 3 to 21 bases  Attachment of Amino Acid to tRNA  tRNA has to be charged to enter the ribosome  A small number of nucleotides in the tRNA and the anti-codon are involved in the recognition by the correct aminoacyl tRNA synthetase  Also by the acceptor stem and the D-loop  Different aminoacyl tRNA synthase  Recognition of tRNA  tRNA synthetase must differentiate between the 20 different amino acids  High fidelity  The acceptor stem is specifically important  A single bp will change specificity  Charging of tRNA  All aminoacyl tRNA synthetases attach an amino acid to a tRNA in two enzymatic steps resulting in the:  Activation of the amino acid - adenylylation  Acyl linkage (charging) between the carboxyl group of the amino acid and the 2’ or 3’ –OH of the adenosine nt on the acceptor arm Step 1: Adenylylation Step 2: Charging of the tRNA  Ribosomes  Ribosome is the machinery that directs the synthesis of proteins  Large and very complex  Composed of two subunits – a large and small subunits  The large subunit contains the peptidyl transferase center  The subunits are named according to their sedimentation velocity - Svedberg Prokaryotic ribosome Eukaryotic Ribosome  Ribosomes  The large and small subunits of the ribosome are made of RNA – ribosomal RNA and ribosomal proteins  The ribosomal RNA (rRNA) is responsible for the key functions of the ribosome  Peptidyl transferase center composed of RNA  The anti-codon loop of the charged tRNA and the codons of the mRNA contact the 16SrRNA and not the ribosomal proteins  Ribosome binding sites  The ribosome has 3 binding sites - A, P and E sites  A -site - aminoacyl tRNA  P- site – peptidyl-tRNA  E -site – tRNA that is released  The ribosome binds two tRNA simultaneously  Steps in Translation  Initiation  Elongation  Termination  The process requires several proteins or factors plus GTP  Initiation of Translation  The ribosome must be recruited to the mRNA  A charged tRNA must be placed into the P-site of the ribosome  The ribosome must be precisely positioned over the start codon  This is important to establish the reading frame for translation of the mRNA  Initiation in Prokaryotes  The mRNAs are initially recruited to the small subunit by base pairing interactions with the RBS and the 16sRNA  The small subunit is positioned on the mRNA such that the start codon is in the P-site when the large subunit joins the complex  During initiation the charged tRNA enters the P-site  The large subunit joins just prior to the formation of the first peptide bond  Initiation in Prokaryotes  The special tRNA known as the initiator tRNA enters the P-site and base pairs with the start codon – AUG (methionine) or GUG (valine)  The initiating tRNA is charged with a modified form methionine – N-formyl methionine  Formyl group attached to its amino group  The charged initiator tRNA is called fMet-tRNAifMet  Not every protein that is made have a formyl group at the amino terminus  The enzyme deformylase removes the formyl group  Most protein in prokaryotes do not start with methionine  Aminopeptidases often remove the terminal methionine as well as one or two additional amino acids  Initiation in Prokaryotes  Catalyzed by three translation initiation factors (Ifs)  IF1, IF2 and IF3  IF1 binds to the A-site  IF2 is a GTPase and interacts with IF1 and reaches over to the P-site to make contact with fMet-tRNAifMet  IF3 binds to the E-site  Initiation in Prokaryotes  With all 3 initiation factors bound the small subunit is prepared to bind to the mRNA and the initiator tRNA  Binding of fMet- tRNAifMet is facilitated by IF2 bound to GTP  Initiation in Prokaryotes  The last step of initiation creates the 70 S initiation complex  This results in the release of IF3  Stimulates the GTPase activity of IF2 to produce IF2+GDP  IF1 and IF2 are released  Now have f-Met in the P- site and the A-site ready to accept the incoming tRNA  Initiation of Translation in Eukaryotes  The ribosomes dissociate into free large and small subunits through the action of eIF3 and eIF1A  eIF2 and eIF5B, GTP binding proteins recruits initiator tRNA  Binding of the initiator tRNA to the small subunit always precedes association with the 5’ capped end of the mRNA  Initiation of Translation in Eukaryotes  The initiator tRNA is charged with methionine  Not N-formyl methionine - Met- tRNAiMet  eIF2-GTP and eIF5B- GTP positions the Met- tRNAiMet in the future P-site of the small subunit  Forming the 43S pre- initiation complex  Initiation of Translation in Eukaryotes  Recognition of the mRNA by the 43S pre-initiation complex begins with the recognition of the 5’ cap on the mRNA by eIF4F  Then joined by eIF4B  eIF4B activates an RNA helicase that unwinds any secondary structures  eIF4F/B recruits the 43S pre-initiation complex to the mRNA  Identification of the start Codon  Once assembled at the 5’- end of the mRNA, the small subunit and the associated factors move along the mRNA 5’ to 3’ in an ATP dependent process driven by eIF4F (RNA helicase) to find the first AUG  The AUG is recognised by the anti-codon of the initiator tRNA  Correct base pairing triggers the release of eIF2 and eIF3  The large subunit can now bind the small subunit  Binding of the large subunit releases the remainder of the eIFs  The Met-tRNAiMet is now at the P-site of the 80S initiating complex  TRANSLATION ELONGATION  The correct aminoacyl-tRNA is loaded in the A-site  Dictated by the A-site codon  Peptide bond formation between the aminoacyl- tRNA in the A-site and the peptidyl-tRNA in the P- site  The peptidyl-tRNA in the A-site is then translocated to the P-site so the A-site can receive another aminoacyl-tRNA  The mechanism of elongation is conserved  TRANSLATION ELONGATION  Requires two elongation factors – EF-Tu and EF-G  Both uses the energy of GTP binding and hydrolysis  EF-Tu-GTP escorts the aminoacyl-tRNA to the ribosome  Masks the aa preventing it from forming peptide bond  EF-Tu-GTP is hydrolysed and is released as EF-Tu- GDP + Pi  Peptide Bond Formation  Peptidyl transferase catalyzes the peptide bond formation  The t-RNA in the P-site is deacetylated and the amino acid linked to the tRNA in the A-site  Peptide bond formation consumes 2 molecules of GTP and 1 molecule of ATP  Peptidyl Transferase  Peptidyl-tRNA in the P site is the ester bond of high energy content that donates its peptidyl group to the amino group of aminoacyl-tRNA bound in the A site of the ribosome  Also involved in the termination reaction of protein synthesis and hydrolyzes the ester bond between the peptide chain and tRNA at the P site of the ribosome  Inhibited by various antibiotics  chloramphenicol, lincomycin, carbomycin (prokaryotes)  cycloheximide (eukaryotes)  Translocation  Translocation requires EF- G  EF-G associates with GTP and binds the A-site after the peptidyl transferase reaction  EF-G-GTP is hydrolyzed to EF-G-GDP and displaces the A-site tRNA into the P- site  The P-site moves to the E- site  The mRNA shifts 3 base pairs  EF-Tu and EF-G are used once for each round of tRNA loading onto the ribosomes, peptide bond formation and translocation  After GTP hydrolysis the GDP is released and bind a new molecule of GTP  TERMINATION OF TRANSLATION  Protein synthesis terminates when the ribosome reaches a stop codon (nonsense codon).  No tRNA binds to a stop codon.  Instead, specific proteins called release factors (RFs) recognize the stop codon and cleave the attached polypeptide from the final tRNA, releasing the finished product.  Following this, the ribosomal subunits dissociate, and the subunits are then free to form new initiation complexes and repeat the process.