Understanding Protein Translation: Key Concepts and Processes
School
Purdue University**We aren't endorsed by this school
Course
BIOL 221
Subject
Biology
Date
Dec 11, 2024
Pages
21
Uploaded by PrivateMeerkatMaster1159
Lecture 15 – Protein TranslationVocabulary3’-poly(A) tail5’-capA siteAminoacyl-tRNA synthetaseAnticodon loopCharged tRNACodonCoupled transcription/translationE siteElongationEF-GEF-TuExonGTPInitiation complexIntronLarge ribosome subunitP sitePeptidyl transferaseReading frameReadthroughRelease factorRibosomal RNA (rRNA)RibosomeShine-Dalgarno sequenceSmall ribosome subunitSpliceosomeStart codon (AUG)Stop codon (UAA, UAG, UGA)Transfer RNA (tRNA)TranslocationTranspeptidationWobbleFrq examples in the ti created review thingy the paper https://quizlet.com/536092111/lectures-14-16-flash-cards/?funnelUUID=4a9cd2b2-6545-4b23-943c-22fa84877e95https://quizlet.com/155916242/bio-221-lecture-15-flash-cards/?funnelUUID=a9ea2dad-dc4c-4ccf-a007-d1362dc73579Learning ObjectivesMrna – template for protein synthesis -Sequence of nucleotides in MRNA determines what amino acids will be in protein TRNA – attach to specific amino acids -Guides amiuno acids to ribosome, directs their addition to growing polypeptide rRNA – Rrna molecules present with many other proteins in ribosome-Necessary for proper ribosome assembly and function Know the process of post-transcriptional modifications that are made to eukaryotic mRNA. Know the process of mRNA splicing that occurs in eukaryotes. Done by what Degradation by what for each + what its recognized by Each mRNA molecule is modified after transcription, so that it can be recognized by the ribosome, and protected by degradation2 structures Added -Formation of 5’ cap structure o5’ 5’ linkage created with phosphates, prevents degradation by nucleases oInverted nucleotide cap (5’ phosphate to 5’ phosphate linkage,) oProteins can recognize it -Formation of poly A tailoPoly a polymerase adds lots of A’s, stabilizes 3’ carbon, prevents degradation oStabilize mRNA, translation proteins can recognize it to start translation
Lecture 15 – Protein TranslationSplicing -Introns (non coding regions ) cut OUT -Exons (coding regions for ribosomes) stays inoSpliceosome recognizes junction between introns and exons, binds to tow junctions, removes intron as a loop After mRNA made , mrna is getting spliced Splicing done by spliceosome -----. All 3 rna structurally the same -MrnaoTemplate for protine synthoSequence of nucleotides in mRna determines what amino acids will be in the protein -TrnaoAttached to specific amino acids Guides amino acids to ribosome and directs their addition into the growing polypeptide -RrnaoRna moelcules present with many other proteins in the ribosome oNecessary for proper ribsome assembly and function Given the structure of a ribosome, and the 5’ end of a translated mRNA, be able to identify the A, P, and E sites.A’ – 3 ‘ endP – middleE - 5 ‘ end
Lecture 15 – Protein TranslationA – acceptor P – petideal E – exit Amino acyl tRNA – adapter that translates a triplet codon in the mRNA to an amino acid in the protein --Ribsome has small and large subunit (LIGHT grey – rRNA ) (in photo) 3 spots where tRNA can dock (APE) Amino acyl trna adapter translates a triplet codon in mRNA to an amino acid in protein Understand the principle of the genetic code and how codons fit. Know the start codon.mRNA is read in groups of three nucleotides, called codons. Start codon – AUG (methionine ) Reading frame – nucleotides read in groups of 3
Lecture 15 – Protein Translation\There are three ways / 3 reading frames to read nucleotides in groups of 3 3 nucleotides is what is being read by the ribosome (64 codes accommodate for 20 amino acids) -Multiple codons for same amino acid -This allows for some mutation to occur with no effect Start codon – AUG (5’ 3’) End codons UAA UAG UGA Know why the ribosome must determine the correct reading from for the mRNA. Know how ribosomes determine where to start reading the mRNA in both prokaryotes and eukaryotes.
Lecture 15 – Protein Translation-Ribosome must start in right are to code correct amino acids, + there are 16 augs in each gene, ribosome must know which 1 reading frame produces the protein Prokaryotes -Shine Dalgarno sequence – (in mRNA) that will base pair with small ribosome subunit. Position start codon in right location in ribosome -Upstream of start codon, but very close -AG rich, small ribosomal subunit can base pair Eukaryote -Lots of untranslated sequence, ribosome finds start codon (AU G) which will set reading frameBoth - Adapter molecules must be used = Trna Anticodon with complementary base pair in tRNA allows mrna to come in and base pair \Know how tRNAs ensure that a mRNA codon is represented by the correct amino acid. Know how aminoacyl-tRNA synthetases ensure that a tRNA is charged with the correct amino acid. Be able to sketch a charged tRNA attached to a mRNA including labeling all 5’ and 3’ ends.
Lecture 15 – Protein TranslationtRNA – series of 3 nucleotides called anti codon. This complementary base pairs with codons Inmrna3’ end where amino acid attached, base = anticodonRibosomal holds mrna, trna binds anticodons to codons in mRNA First two nucleotides Last nucleotide in anticodon Lower than 64 trna, 1 can recognize many codons and deliver same amino acid TRNA ensures mRNA codon is repped by correct amino acid thu tRna anticodon binding to its specific codon on mRNA.-Diff aminoacyl tRNA synthetase for each tRNA, and it recognizes which amino acid gets attached to which tRNA oIs has 2 active sites, 1 for anticodon on tRNA, other for recognizing amino acid R group. oIt catalyzes a covalent bond between them, each one recognizes a specific anticodon and r Group Arnet 61 trnas, wobble. One trna can bp with more than one codon First nucleotide in anticodon, pairs with last bp in codon. Aasyntehstase – recognize anticodon on trna and r group on aminoacid, covalently bind to both.
Lecture 15 – Protein Translation-----E k Anti codon / codon pairing – checked aminoacyl trba synthase Ensures amino acid put on right trna Mrna bind to trna anticodon (which contains amino acid) This amino acid is taken to ribosome, added to polypeptide chainTrna repeats process with new mRNA tRNA binds to mRNA codon by complementary base pairing 1.Initiation factors and GTP bind to 30s ribosomal unit2.Intiator trna and mRNA bind to 30s ribosomal unit 3.50s ribosomal subunit becomes bound to 30s initiation complex 4.r\when ribosome is assambeld, tRNA carrying the first amino acid is paried wit hstart codon on mRNA . tRNA carrying second amino acid approaches
Lecture 15 – Protein Translation5.– 6.Three bases in mRNA recognized by tRNA is called codon Process consumes energy Elongation factor with charge ef – tu = means is bonded to mRNA Charge trna goes to A spot, moves over, uncharged empty trna in that spot will leave in Eand be recycle to amino acyl Translocation reaction cataluzed with ef-g insinuates itself into a site, pushing tRNAs to next sites
Lecture 15 – Protein TranslationGiven a muted animation, be able to narrate your way through the animation process of protein synthesis. (slides 36 ) are good. Remember release factors binding to a site at end
Lecture 15 – Protein TranslationInitiation Shine Dalgarno binds to small ribosome start unit-Places it in P site of ribosome Initiator TRNA -Anti codon (UAC, for AUG) -First amino acid fmet -When tRNA base pairs to start codon, allows large ribosomal subunit to join Ape sites now presentA – next codon available (amino acyl site/acceptor) -tRNA will use its anticodon to pair with A siteP – holds growing peptide chainE – exit site -tRNA’s that have lost amino acid to growing chain exit
Lecture 15 – Protein TranslationShine dalg binds to small ribosomal start unitInitiator trna – (anticodon uac) binds to psite with Fmet amino acid Large ribosomal subunit joins Elongate Trna binds to A site, polypeptide (peptide bond catalyzed) transferred to a site, trna leaves to E site Ef tu ensures proper anticodon with codon, if wrong trna ef Tu removes the trna Ef g – translacation (moves trnas) Gtp used for ef tu and ef G Transpeptidation – peptidyl transferase Translcaotion – mRNA movement
Lecture 15 – Protein TranslationNext tRNA coming in is AAU in this case Ribsome catalyzes peptide bond with tRNA in A and P sites Three bases in mRNA recognized by trna are codon Translation factor Ef TU – makes sure we have appropriate anticodon with codon, -costs GTp (3 per amino acid) -if anticodon/codon don’t match, EF TU kicks out faulty tRNA Ef-G – moves tRNA during translocation Transpeptidation - peptidyl transferase (peptide bond formation) Translocation – sliding of ribsome/mRNA moved so a site becomes open each time
Lecture 15 – Protein TranslationElongation Incoming tRNA with Ef-tu binds to A siteP site – growing chain of amino acids moves thru exit tunnel, emerges other sideAs ribosome translaties and slides, the trna will go form Ape-EFG allows tRNA to slide form a to P site -trna in P site will become uncharged, goes to E site to exit, then will be recharge by amino acyl trna synthetase Termination-tRNAs cant match the stop codon -ribosome will Stall -release factor binds to codon in A site instead of tRNA and causes ribosome to fall apart -1 / 21 chance of stop codon after first one Overall below
Lecture 15 – Protein TranslationIntatior tRNA binds to P site
Lecture 15 – Protein TranslationExplain why occasional “readthrough” of a stop codon does not damage a cell.Sometimes a tRNA does recognized a stop codon, but its ok as the sequence beyond stop is random, and contains other stop codons Be able to sketch the coupled processes of transcription and translation, labeling all 5’ and 3’ ends, the promoter, Shine-Dalgarno sequence, start codon, stop codon, and transcription terminator.Bacterial DNA in ctoplasm, as mRNA made, ribosomes are readily avvalible -They can make proteins as mRNA is created Starts at promoter. mRNA is being madeWhen enough length, ribosome binds to shine Dalgarno
Lecture 15 – Protein TranslationWill keep going down to terminator sequence
Lecture 15 – Protein TranslationInitiation
Lecture 15 – Protein TranslationElongation/termination
Lecture 15 – Protein TranslationKnow three ways antibiotics kill bacteria by inhibiting protein synthesis.Many antibiotics target protein synthesis We target cell walls and ribosomes -Block initiation -Block elongation -Prevent transpeptidation -Block translocation -Inhibit isoleucine tRNA synthetase -ST CC EM (photo below is good for this one)
Lecture 15 – Protein TranslationSTCC EM STCC EE Know how DNA mutations can lead to mutant proteins and changes in cell functions.4 types Any change in codoing region of a gene can affect h bonding in protein alter protein function(point mutation alter one codon, changes amino acids) (insert/delete mutation change reading frame) ]](promoter alteration can lead to increased or decreased transcription (change number of copies of protein in the cell) If the protein is an enzyme, its function can be lost, resulting in a mutant metabolic phenotype Any change in DNA -change codons in mRNA -One nucleotide= opoint mutation (changes one amino acid, can be negligible or malfunction ) -Insert /deletion oChange reading frame , read wrong grouping of 3 (wrong amino acids in protein, struc and function altered ) Mutations can happen in promoter regions (sigma factor binds) -Can increase or decrease level of protein, can have phenotypic effect on cell -Enzymes can have metabolic defects
Lecture 15 – Protein TranslationRibsome moves from 5; to 3’ A site = wobble site (3’)