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Beadle and Tatum: bombarded bread mold with xrays, gave the bread mold minimal medium supplemented with different intermediates in the arginine pathway; they determined the metabolic pathway for arginine synthesis; one enzyme one gene (now known as one enzyme one polypeptide) Garrod: analyzed urine and discovered alkaphenoturia (sp); discovered that an enzyme was missing in the metabolic pathway, if it was it would turn urine dark, dark deposits in ears, eyes, along spine What composes RNA, protein, DNA? RNA (made of U, A, C, G and a ribose sugar/phosphate backbone); proteins made of amino acids with a specific R group, there are 20 different amino acids; DNA made of deoxyribose sugar/phosphate backbone T, A, C, G) What is a codon? Anticodon? Where are they found? Triplet code of nucleotides found on the mRNA that codes for a specific amino acid; anticodon is the complementary nucleotide sequence that coincides with a “specific” codon on the mRNA , it is attached to the tRNA that carries a specific amino acid How do you interpret a codon wheel or chart? What are the differences, if any, for the genetic code for organisms? Prokaryotes have circular DNA; some of the codon sequences do not code for the same amino acid and can be substituted (most codons code for the same amino acid but there can be subtle differences in this between organisms) Mechanism for transcription in eukaryotic vs prokaryotic? Eukaryotes require transcription factors that allow RNA polymerase to bind to the promoter (TATA box); occurs in nucleus; have introns and exons; spliceosomes remove the introns; mRNA gets processed to add 5’cap and poly A tail which helps it to get out of the nucleus; repressors can bind to the activator and stop transcription, or an inducer can bind to the activator and start the pathway Prokaryotes: happens in cytoplasm, has exons but no introns, no TATA box, no transcription factors, RNA polymerase still binds to transcribe the DNA, mRNA not processed (no 5’cap or poly A tail) because translated immediately; Mechanisn for translation in eukaryotic vs prokaryotic? Both use ribosomes, both happen in cytoplasm, See ribosomes, tRNA below Primary transcript? The original mRNA transcript of the DNA before the introns are cut out, before the 5’ cap is added, and the 3’ poly A tail is added Processed transcript? mRNA transcript that has had the introns removed by spliceosomes, the spliceosomes splice the exons back together; 5’ cap is added (it is a modified guanine and 3 phosphates); the Poly A tail is added to the 3’ end; the 5’ cap and poly A tail prevent degradation of the mRNA, helps the mRNA to leave the nucleus, and helps the ribosome attach for translation Alternative splicing? How multiple polypeptides can be made from the same mRNA transcript depending on which introns are removed from the transcript Introns? Exons? UTRs? Introns are noncoding regions that get removed from the primary transcript, Exons are the coding regions of the nucleotide sequence; UTR noncoding region that remains in the processed mRNA transcript thought to be regulatory Ribosomal RNA? What is it made of? How does it work? Made of large subunit (RNA and proteins) and small subunit (RNA); contains the E site, P site, A site; mRNA attaches to the mRNA site on the small subunit of the ribosome; tRNA is brought to the mRNA by the aminoacyl synthetase and brings the appropriate amino acid to the mRNA (matches the anticodon site on the tRNA to the codon site of the mRNA); the tRNA is delivered to the A site (the enzyme is then released to get/charge another tRNA); the tRNA then moves over to the P site and the amino acid it carries is attached to the growing polpeptide chain; the tRNA is then moved to the E site and ejected from the ribosome to go off and be charged again Transfer RNA? How does its 3d shape occur? How does it work? Brings the appropriate amino acid to the ribosome; usually RNA is linear, 3D shape occurs when the RNA folds back on itself and finds complementary sequences and hydrogen bonds to itself; it has two binding sites- one site holds the amino acid, the other site has the anticodon that will bind to the codon region of an mRNA in a ribosome mRNA? Messenger RNA, a complementary strand to DNA that is processed and allowed to leave the nucleus of the cell Where does transcription occur? Translation? In prokaryotes? In eukaryotes transcription occurs in the nucleus, translation occurs in the cytoplasm; prokaryotes do not have a nucleus so transcription/translation occurs simultaneously in the cytoplasm DNA to mRNA to amino acid (be able to decode, transcribe and translate) see codon wheel above and practice Gatcatctgatc (DNA sequence) Cuaguagacuag (RNA sequence) Cua/gua/gac/uag establish reading frame Look up codon on codon chart Leu-val-asp-stop codon Order of protein synthesis? DNA is transcribed to RNA; RNA is translated to protein What does wobble mean and what does it allow? It allows for flexibility of the tRNA at the 3rd base of the codon; it allows tRNA to bind to more than one codon and deliver the correct amino acid (there are 45 different tRNAs that can recognize 60 different codons) What is a reading frame? The 3 nucleotides that are read to correctly create a codon What can cause reading frame shifts? Deletions, insertions can cause reading frame shifts What other types of mutations can occur? Base pair substitutions; missense, nonsense, silent mutations; translocations, inversions Operon model? An operon contains the operator, the promoter, the genes that are going to be expressed, RNA polymerase; shows how DNA is transcribed in prokarytoes and some eukaryotes; RNA polymerase attaches to the promoter, if no repressor the RNA polymerase will untwist the DNA and creates RNA nucleotides, once the DNA is transcribed the RNA polymerase “pops off” of the DNA and the transcription complex comes apart Example: trp operon, lac operon Lac operon: lac pathway is inducible operon that is always turned off but can be turned on by allolactase (enzyme that inactivates the repressor by binding to the repressor and changes the shape of the repressor so it can’t bind to the operator and allows the transcription pathway to continue) repressible operon? The pathway is usually on but can be slowed/stopped by the repressor binding to the operator Inducible operon? Pathway is usually off but can be started by the repressor being inactivated Regulatory genes? Genes that code for a protein, such as a repressor (or an enhancer) that controls the transcription of another gene or group of genes Promoter? A specific nucleotide sequence in the DNA that binds RNA polymerase, positioning it to start transcribing RNA at the appropriate place Repressor? A protein that inhibits gene transcription; in prokaryotes repressors bind to the DNA in or near the promoter; in eukaryotes repressors can bind to the control elements within enhancers, to activators, or to other proteins in a way that blocks activators from binding to DNA Inducer? a specific small molecule that binds to a bacterial repressor protein and changes the repressor’s shape so that it cannot bind to the operator thus switching the operon on. Enhancer? A segment of eukaryotic DNA containing multiple control elements usually located far from the gene whose transcription it regulates Activators? Proteins that bind to certain mediator proteins and general transcription factors helping them form an active transcription initiation complex on the promoter Nonsense? When a point mutation changes the original codon to code for a stop codon; no polypeptide can be made Missense? a point mutation that changes an amino acid in the polypeptide chain Silent? A point mutation that doesn’t alter the amino acid that is added to the chain (changes the codon for the same amino acid) How much of the genome codes for genes? What does the rest do? 1.5% actually codes for genes; used to be considered “junk” but being found to code for regulatory aspects of protein synthesis Epigenetics? How environmental factors can alter gene expression by affecting the condensing of chromatin and how the transcription machinery can “fit” onto the chromatin to start transcription; REMEMBER: DNA nucleotides can not be altered but the epigenome can; methy groups can attach and condense the chromatin; if methyl groups are taken off the chromatin relaxes; addition of an acetyl group does the opposite. siRNA ? microRNA? siRNA (small interfering RNA) and microRNA (smaller than small siRNA) work the same; they are sequences of RNA that attach to the mRNA and cause hairpin loops; if the microRNA or siRNA are a perfect complement to the mRNA, the mRNA will be completely destroyed by the dicer enzyme; if a siRNA/microRNA is a close match then translation is slowed down totipotent? a cell that can still become any type of cell (stem cell) Pluripotent? A cell that has been “determined” to be a certain type of cell but hasn’t started differentiation Determined? An “apprentice cell” that knows what type of cell it will be but hasn’t started to “change” into that cell type Differentiated? A cell that becomes specialized. A “master”cell Transcription factors? Proteins that bind the promoter and enable RNA polymerase to bind (there are atleast 1000 transcription factors that are specific for the particular sequences that code for specific gene expression) Egg polarity genes? Help to determine body plan; sets up anterior/posterior part of embryo and dorsal/ventral axis (example was bicoid gene) Homeotic genes? Control pattern formation in the late embryo; highly conserved sequence in a lot of eukaryotes Post transcriptional control? mRNA degradation by the removal of the poly A tail, the 5’ cap; microRNA/siRNA slowing or stopping translation; primary transcript of mRNA can be alternatively spliced to synthesize different proteins; regulatory protein can bind to the UTR region of the mRNA and temporarily/permanently stop translation initiation