Objectives for Exam 4 PCB 3063 Summer 2012 Chapter 13 Translation and Proteins Q Know the major components of the translation apparatus mRNA tRNAs and ribosomes know what their roles are in translation Translation of mRNA the polymerization of AA s into polypeptide chains occurs only in association with ribosomes think of as workbenches tRNAs bring AA s to the ribosome are adaptor molecule that associate codons with specific AA s ribosomes complexes of proteins and RNAs rRNA Q Know that the ribosome has two subunits large and small each of which is composed of one or more RNA molecules and a large number of proteins Ribosomal Structure o consists of 2 subunits each of which consist of one or more molecules of rRNA and an array of ribosomal proteins o eukaryotic ribosomes are larger than prokaryotic ribosomes Q that carry out this reaction and what their specificity is they recognize and join the correct tRNA with the correct amino acid Before translation can proceed the tRNA molecules must be chemically linked to their repective AA s Aminoacyl tRNA synthases direct the activation process link a tRNA molecule and an AA which is now a charged tRNA o are very specific because they only recognize one AA and the subset of corresponding tRNAs Only 20 different synthases Q Be able to describe in general terms the three steps in protein synthesis in addition to tRNA charging chain initiation chain elongation chain termination Know the roles in translation of the A site and the P site on the ribosome Initiation 1 small subunit binds to the mRNA at the position of the start codon 2 initiator tRNA fmet binds to mRNA codon in the P site of the ribosome initation complex large subunit binds to the complex elongation factors bind to tRNA facilitating entry into the A site 3 Chain Elongation 1 charged initiator tRNA binds to P site once the P and A sites are assembled with the mRNA after initiation the next charged tRNA binds to the A site 2 3 a peptide bond is formed and tRNA shifts from the A site to the P site 4 Ribosome slides 3 bases down the mRNA Chain Termination 1 when the stop codon is reached termination factors bind cleave the polypeptide from the last tRNA and ribosome mRNA complex falls apart Q Understand how the correct start codon is identified during the initiation of translation in prokaryotes and in eukaryotes How is the reading frame set how is the correct AUG recognized and distinguished from internal AUG codons Prokaryotes o tRNAimet is different than tRNAmet o methionine attached to tRNAimet is formylated fmet o only tRNAimet can enter the P site directly Shine Dalgarno sequence o serves as a ribosome binding site in the leader sequence of prokaryotic genes o 7 10 bases before the start codon o AGGAGG AUG o The ribosome binds to the mRNA at the SD sequence o also have a different tRNAmet for initiation and for internal AUG met no o 5 guanosine cap needed for initiation o the small subunit of the ribosome binds to the cap and slides down to the first Eukaryotes formylated AUG Kozak Sequence o similar to Shine Dalgarno sequence usually surrounds the first AUG o short nucleotide sequence adjacent to the initiation codon that is recognized as the translational start site Q Know the importance of the Shine Dalgarno sequence of prokaryotes what it does where it is located and the Kozak sequence in eukaryotes Q Understand the concept of reading frame and be able to identify all three reading frames in a piece of RNA SEE ABOVE SEE ABOVE Q Know the experiments done with the fungus Neurospora in which auxotrophic mutants were isolated and characterized and from which it was concluded that each gene contains the information for making one polypeptide o Induced mutations and produced strains that had genetic blocks of reactions essential to the growth of the organism o The used X rays or UV radiation to induce mutations in the mold o They then tested the individual spores on minimal medium if they failed to grow on minimal medium then the spores had acquired a nutritional mutation o To identify the mutant type the mutant strains were then tested on a series of different minimal media each containing groups of supplements and eventually on media with single purines pyrimidines AA s until one specific supplement that permitted growth was found o Beadle and Tatum thought that the supplement that restored growth was the molecule that could not be synthesized by the mutant strain o They provided experimental evidence for their hypothesis that one gene encodes one enzyme Q Be able to solve problems in which the order of enzymes in a biochemical pathway is determined using auxotrophic mutants grown on minimal medium plus intermediates in the pathway SEE HOMEWORK PROBLEMS AND ANSWERS Q Understand what genetic complementation means and how genetic crosses are used to identify alleles o Complementation crosses between two different mutants corrects the phenotype Q Be able to explain using specific examples why some alleles are recessive some exhibit partial or incomplete dominance and why some are dominant o Recessive alleles result in the loss of function of the protein they code for o ex sickle cell anemia defective gene o Dominant mutations structure of one polypeptide is altered in protein with multiple subunits each subunit is coded for by a separate gene o ex Marfans syndrome is dominant affects fibrillin which regulates the elasticity of collagen Chapter 14 Mutations and DNA Repair Q Know the difference between spontaneous and induced mutations between somatic and gametic mutations o Mutation change in DNA base sequence that is transmitted through cell division o Gene mutations small changes in base sequence substitutions single base insertions etc this is what we re referring to in class most of the time o Chromosome mutations large deletions translocations inversion we don t need to worry about these just know the difference o Gametic mutations mutations in the germline will be passed on inherited o Somatic mutations can lead to cancers etc but will not be passed on occur in disorders pg 286 any cell except germ cells o Base substitutions point mutation may result in the creation of a new triplet that codes for a different AA in the protein product o Base transitions switch one purine for another A with G or one pyrimidine with another pyrimidine C with T o Base transversions purine for pyrimidine or vice versa o base trasitions are 5 10x more common o Spontaneous mutations due to
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