BIOL 213 1st Edition Lecture 16 Outline of Last Lecture I. DNA mismatch repairII. Chemical damage to DNAa. Leads to mutationsIII. General mechanisms of DNA repaira. Recognition and excisionb. Replacementc. Joining IV. Different methods of replacementa. Recombinationi. Homologousii. Non-homologous1. Transposons V. Viruses use non-homologous recombination when inserting their DNA into the host’s DNAa. Retroviruses VI. Transcriptiona. In prokaryotes vs in eukaryotesOutline of Current Lecture I. Splicinga. Introns and exonsII. Comparing eukaryotes and prokaryotes processes These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.III. Translationa. Codonsb. It takes place at a ribosomec. Initiation and terminationIV. Protein synthesis and longevity are regulatedCurrent LectureI. Splicinga. mRNA has to be processed before it can leave the nucleusb. Splicesosomes cut out the introns (non-coding sequence) and link the exons (coding regions) togetheri. There are specific sequences that identify an intron-exon boundaryii. Sequences required:1. 5’---AGGURAGU---[MORE RNA]---YURAC---[MORE RNA]---YYYYYYYYNCAGG---3’2. Bold = exon3. Italics = introns 4. R = A or G Y = C or U5. The spliceosome will cut the intron out and the exon sequence will be: ---AGG--- c. The introns form a lariat (a loop)i. One of the ends bonds to the 2’ –OH of an adenine nucleotide through dehydration synthesisd. Alternative splicing can lead to different proteinsi. Different exons from the same gene will be joined together in different orders to create different genesii. Each unique gene has its own unique spliceosomeiii. This is important for the different kinds of cells – if all cells made the same amount of all proteins, they wouldn’t be as specialized as they are1. Ex: muscle cells make more actin and myosin than neuronse. Errors can lead to nonfunctional proteinsi. Exons can be skippedii. Only pieces of exons can be splicedf. In order to leave the nucleus, a processed mRNA has to have specific proteins bonded to it:i. A polyA-binding proteinii. A cap-binding complexiii. A nuclear transport receptorII. Comparing eukaryotes and prokaryotes processesa. Prokaryotic is very simplei. DNA is synthesized in the cytosol ii. RNA is transcribed from DNA in the cytosoliii. RNA is translated to proteins in the cytosoliv. There is no mRNA processing – no 3’ cap; no polyA tail; no splicing; no introns or exons (all prokaryotic DNA codes for genes)b. Eukaryotic is a lot more complexi. DNA is synthesized in the nucleusii. RNA is transcribed from DNA in the nucleusiii. mRNA is processed in the nucleus before it can leave to the cytosol1. a 5’ cap is added2. A polyA tail at the 3’ end is added3. Introns are cut out and the exons are spliced together4. Appropriate proteins are attached to the mRNAiv. mRNA moves through the pores in the nuclear membranev. mRNA is translated in the cytosolIII. Translationa. It’s very different from transcription in that it isn’t a direct conversion of nucleotidesi. There are 4 different nucleotides and 20 different amino acids –ii. If it were a direct 1-to-1 conversion, there would only be 4 amino acidsb. Every 3 nucleotides (a codon) code for one amino acidi. There are 43 = 64 possible comibinationsii. But there are only 20 amino acids, so multiple codons can code for the same amino acidc. This was tested by using synthetic mRNA and experimenting with it to determine which sequence of nucleotides codes for which amino acidsd. Multiple codons can code for the same amino acid due to third position “wobble”i. The piece of the tRNA that has the anticodon is a flattened end of a loopii. The first nucleotide of the anticodon is located on the corner, so it’s not as specific as the second and it because it can move around1. mRNA and tRNA are read from 5’  3’ so the third nucleotide of the codon corresponds to the first nucleotide of the anticodoniii. This allows different nucleotides to bond in the third position of the codoniv. For example: alanine is code by GCA, GCC, GCG, GCU1. The first two are the same; it differs in the third nucleotidee. Codons to memorize:i. Start = methionine: AUGii. Stop = UAA, UAG, UGAf. Different initiation sites leads to different proteinsi. If you have an mRNA strand of 5’…GCGAAUGCU…3’, you could have codon sequences of:1. …GCG AAU GCU… or2. …G CGA AUG CU… or3. …GC GAA UGC U… or4. Each of which codes for different amino acid sequencesg. Each amino acid is linked to the tRNA that has its corresponding anticodon by theenzyme tRNA synthetase i. Each amino acid has its own tRNA synthetaseii. It is linked by a covalent bond1. ATP is used to create this bondiii. This bond holds a lot of energy and provides all the energy needed when linking the amino acids together to create the proteinh. Protein synthesis takes place at the ribosomei. There are two ribosomal subunits, a small and a large1. They come together near the 5’ end of the mRNA2. The small matches the tRNA with codons3. The large catalyzes the formation of peptide bondsa. Remember no energy input is required because it’s alreadyin the bond between the tRNA and the amino acidii. There are three binding sites in a ribosome1. A site – first a. This is the initial point where the tRNA matches up with the mRNA; b. The amino acid from the tRNA in the P site is bonded to the amino acid of the tRNA in the A site2. P site – second a. This is a place holder for the tRNA that transfers its amino acid3. E site – thirda. This is the site where the tRNA gets ejected from the ribosomeiii. The tRNA with methionine (the start codon) binds to the P site and then passes its amino acid to the tRNA that binds to the A site1. This is the only tRNA that can bind directly to the P site2. This binding is assisted by proteins called translation initiation factorsiv. There is now a tRNA in the P site and A sitev. The large subunit moves over one codon so that there is a tRNA in the E site and P sitevi. The small subunit moves over to match the largervii. The tRNA in the E site is ejected and a new tRNA binds to the now open Asite1. The ejected tRNA gets another amino acid attached to it by tRNA synthetase i. Translation initiationi. In eukaryotes1. The small subunit and translation initiation factors recognize the 5’cap and bind to the 5’ end of the mRNA2. They move along the