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TRANSCRIPTION-TRANSLATION PROTEIN SYNTHESIS BEGINS Somethin’ weird In the neighbourhood Who you gonna’ call? Protein Synthesis History • Garrod (1902) one gene one enzyme hypothesis • Beadle and Tatum(1935-1944) one gene one polypeptide hypothesis • Vernon Ingram gene specifies the kind and location of each amino acid in a polypeptide chain. • RNA links DNA to protein sequence • Crick’s central dogma Early 20th century geneticists were faced with several problems: 1. What are genes? 2. How do genes work? 3. What produces the unique phenotype associated with a specific allele Yea, 20th century scientists We’re in the 21st century you know. Back off man, we’re scientists The ONE GENE ONE PROTEIN HYPOTHESIS. Garrod a physician 1909 postulates Hey, what kind of urine sample is this? It’s not like the others with the amber colour. How can the urine be black? I want to investigate that. Black Urine. That’s when you call.... I tested the urine and I found it contained alkapton. Alkapton turns black on contact with air and it was causing the black urine colour. I furthered the study and discovered the chemical was in the blood of the patient as well. This led me to believe that it was an inborn (genetic) error in metabolism. By the way, I determined it was an autosomal recessive gene My hypotheses were 1.Individuals with alkaptonuria have a defective gene, making them unable to produce the correct alkaptonmetabolizing enzyme that most people possess. 2. The enzyme is produced as a result of hereditary material. Defective hereditary material leads to defective enzyme. A Enzyme 1 B Enzyme 2 C Enzyme 3 B Initial reactant Intermediate metabolites B B B Final product EXAMPLE If enzyme 2 is defective, substance B will accumulate One gene, one enzyme hypothesis - A single gene controls the specificity and activity of each enzyme in a given metabolic pathway MEANING- mutation of this gene changes the ability to the cell to carry out a particular reaction, thus disrupting the pathway. D Next on the scene is Beadle and Tatum They demonstrated experimentally Garrod’s hypothesis to the one gene one enzyme and changed it to the one gene one polypeptide Hypothesis. when TATUM Shhh. Don’t say BEADLE S fungus, most battle with paddles people associate in a puddle, it with my athlete’s foot they call it a tweetle beetle puddle paddle battle. AND... When beetles... We used fungus in our experiment! The Beadle and Tatum EXP is great! STEP 1 CAUSE the MUTATION STEP 2 Take samples that will not grow on minimal medium like the normal Neurospora, and grow them on enriched These are the mutants- but for what protein? STEP 3 Switch cultures from enriched medium to minimal medium with a particular supplement. If you have growth of the culture, this identifies the mutant strains that require the stated nutrient One gene one polypeptide Revision of the earlier one gene one enzyme hypothesis . Some proteins are made up of multiple polypeptide chains and these are coded by separate genes. MEANING- a mutation in a gene coding for a specific polypeptide can alter the ability of the resultant protein to function. This can result in an altered phenotype of the individual! Guys these are not enzymes affected by mutations. These are one of the polypeptides that makes up the protein hemoglobin. Lets go see my friend Vernon! Just look at those filthy mutated blood cells I’d recognize those sickle shaped blood cells anywhere! Isn’t this guy too old to work with us? Relax, the guy is a genius. He is considered the FATHER OF MOLECULAR MEDICINE. He figured out that sickle cell anemia is caused by a gene with a defect for a single amino acid He’s right , you know. All of our advances stand on the foundations of these dedicated scientists work; especially the old ones. Dr. Vernon Ingram linked a human hereditary abnormality(sickle cell anemia) to a single alteration of an amino sequence in the protein. This is a single defect in the genetic code that is expressed by the individual. Mutated Sickle gene Normal gene Remember me? I helped support this effort Dr. Ingram and others determined that the hemoglobin molecule of sickle cell disorder was due to the substitution of glutamic acid in position 6 of the β chain. Ingram used electrophoresis and chromatography to show the difference between the normal and mutated gene . This is the machine for gas chromatography. (not paper chromatography like we did) It separates out chemicals based on molecular character. It’s a highly sought skill if you can work one of these. CSI kinda stuff! Electrophoresis Check us out Get trained here in gas chromatography. We actually have the machines you can train on, unlike some local universities! A enzyme1 B enzyme2 C enzyme3 D The above diagram corresponds to a polypeptide pathway. A, B,C,D are all represented in the graph below and your job is to determine which colour each of the letter boxes is from the experimental evidence of different mutants of bacteria growing on different media. MUTANT GROW ON RED GROW ON BLUE GROW ON PURPLE GROW ON BLACK 1 GROWTH NO GROWTH NO GROWTH GROWTH 2 GROWTH NO GROWTH GROWTH GROWTH 3 NO GROWTH NO GROWTH NO GROWTH GROWTH Mutants will lack enzymes 1 2 3? Red must be third as 2 varieties grow on it Blue must be first as none of the mutants can grow on it. You need all three enzymes to get to D Purple must be second as only1 variety grows on it Black must be last as all varieties survive on it. You don’t need enzymes if it is given RNA is the link between the DNA and the protein sequence Scientists for some time had suspected such a link between DNA and proteins Evidence pointed to the link being the RNA molecule as RNA is found is not restricted to the nucleus like the DNA. RNA is also found on the ribosomes in the Endoplasmic reticulum Now it’s time for me to discuss my Central Dogma It’s not so bad. Think of Einstein. He did his best stuff when he was a patent clerk. You don’t have to be like Francis Crick and work at Cambridge University. It’s a major disgrace losing our positions at the university. Forget MIT or Stanford now. Personally, I liked working for the university! They gave us money and facilities. We didn't have to produce anything. You've never been out of college. You don't know what it's like out there! I've worked in the private sector... they expect results The central dogma states information flow is from DNA to RNA via transcription and thence to protein via translation. DID YOU KNOW? Only about 3% of the human genome is both transcribed and translated. This means a vast amount of the DNA is not apparently used!! THE SECRET CODE OF DNA Transcription makes a Translation makes polypeptides copy of DNA on the mRNA from the mRNA codes. In prokaryotes, shown here, the promoter is needed to bind RNA polymerase The side of the DNA the promoter (P) is on is the same side of the DNA that will act as the template strand. I am the end! T P The terminator(T) signals the end of transcription You can see from the diagram that the promoter is upstream and the terminator downstream. The yellow arrow indicates the copy direction. DNA has code which is like a language. It stores vast amounts of information utilizing only 4 different chemicals ATCG for short. 1.DNA is transcribed to mRNA which is the chemical that is to be read like a language. It has the letters AUCG instead of our 26 letter alphabet 2. You have to know what direction to read in. English is right to left but Arabic is left to right. RNA is read in a 5’ to 3’ direction 3.Words in languages are separated by spaces. There are no spaces in RNA so each “word” is called a codon and is three nucleotides in length. 4.Sentences begin with a capital to indicate a start. In codon language starts are indicated by AUG which is the code that translates to the amino acid methionine 5. Sentences indicate a completion of information with a period. RNA indicates the end of the code with a stop signal, which are specific codons. The secret code of DNA involves the use of 4 different letters in codons that are 3 nucleotides long. These must code for the 20 amino acids. Why aren’t the codons only two long or four long. Why are they always 3 nucleotides long? Mathematically in order to determine how many different codes can be made using only two letter code you would get 42= 16 which is not enough to code for the 20 amino acids. If you go to the next smallest letter (3 letters) code you get 43 = 64. This will accommodate all 20 amino acids, allow for some stop signals and allow for some repetition of letters that will form the same amino acid . Ie – Allows for some errors without errors in the amino acid determined in TRANSLATION. This is the strand that you use to make the mRNA copy. It is called the template strand or antisense strand. You can see from this layout that you are ready to translate the RNA code. Remember the codons are always three nucleotides long. The secret code must be decoded utilizing the mRNA codon chart on the next slide. met pro leu asp arg stop Once you hit the stop codon you STOP and the polypeptide is finished even though there may mo more codons Follow the coloured boxes to translate the codons We noticed that for many of the codons, they code for the same amino acid. Most allow for a mistake on the third nucleotide of the codon. WOBBLE ANTICODONS CAN PAIR WITH MORE THAN ONE CODON AS A RESULT!! The Wobble hypothesis states that there are synonymous codons for a given amino acid usually differing in their third base. WOBBLE enables the third nucleotide of some tRNA anticodons to pair with more than one kind of base in the codon. EUKARYOTIC PROTEIN SYNTHESIS promoter TATA In eukaryotes, the promoter, several transcription factors and usually a TATA box are needed to initiate transcription complex Problem here. You have a brain and can use the chart to decipher the codons; the cells don’t. So how do they do it? The first step, called TRANSCRIPTION, is initiated in the nucleus of eukaryotic cells. Primary transcript OR transcription unit produced by RNA polymerase II 3’ 5’ As RNA polymerase II copies it is called ELONGATION Introns removal RNA polym erase Post transcriptional modification removes introns leaving exons which are joined together. Spliceosomes remove introns with snRNA snRNP Exons spliced together 5’ cap Coding part Poly A tail During transcription the 5” end is capped with modified G the 3’ end is modified with a poly A tail. Note: RNA polymerase II is used in EUKARYOTIC transcription. RNA polymerase I and II are not translated The mRNA transcript now leaves the nucleus through the nuclear pores and enters the cytoplasm for the TRANSLATION STEP EUKARYOTIC TRANSCRIPTION TERMS: Upstream begins downstream at end Remember that in the transcription process, the RNA polymerase only copies the template(antisense) strand to mRNA in a 5’ to 3’ direction. You may notice, however, that the mRNA strand is almost the same as the DNA copy (sense) strand only with all the T replaced by U. SPECIAL NOTE: TRANSCRIPTION Eukaryotes have three different RNA polymerases for transcription RNA polymerase I makes ribosomal RNA RNA polymerase II makes mRNA genes that code for protein RNA polymerase III makes transfer RNA Transcription occurs one nucleotide at a time RNA polymerase does NOT proofread TRANSLATION – THE PLAYERS ala met gly thr val In the cytoplasm where translation takes place, All these RNA molecules are synthesized in the there are 3 different types of RNA. Messenger mRNA with the codons, ribosomal rRNA which together with a little protein makes the ribosome, and transfer tRNA with the anticodons and attached amino acids nucleus by RNA polymerase. Nucleoli show high transcription 40s 60s This ribosome is a eukaryotic variety called 80s. It has a 40s and 60 s subunit (sorry they don’t add up) DID YOU KNOW? We have our own DNA and ribosomes, but both are more similar to prokaryotic cells! The mitochondria and chloroplasts have their own ribosomes and these are 70s, similar to those found in bacteria of today. This supports the endosymbiotic theory of the origin of these organelles. Antibiotics like tetracycline and streptomycin target the bacterial ribosomes 70s but do not harm the 80s variety of eukaryotes. The 70s ribosomes of mitochondria are not harmed because they are inside the cell protected by a double membrane. Recent evidence concludes that the functional part of the ribosomes are made of RNA. This again supports the concept of the ribozyme- RNA acting as both an enzyme to catalyze a reaction and also storage of hereditary information. Listen, you just have to check out my presentation on origin of First Cells in the evolution section. Learn about RNA evolution there! The anticodon will match the codon sequence and thus direct this tRNA carrying the amino acid glutamic acid to the correct region to be attached Glutamic acid is electrically charged. It’s obvious because it’s an ACID! Acids ionize glu Each transfer RNA becomes charged with the appropriate amino acid by the enzyme aminoacyl synthetase. In the process an ATP is used up and converted NOT to ADP but rather AMP!!! The ribosome has three attachment points for the tRNA and forms the peptide bonds. The A site is the ATTACHMENT site for the tRNA. E site P site A site The P site is the PEPTIDE site and it is where the peptide bonds are formed between the amino acids The E site is the EXIT site, where the tRNA is released to be recharged and recycled. Note : Two of these these names are simplified for better understanding . A site is aminoacyl binding site , P site is peptidyl binding site TRANSLATION met pro leu asp arg TRANSLATION is accomplished by a method similar to that animated here. One omission is that each tRNA must be shown binding only when the ribosome moves and makes the A site available. GTP is hydrolyzed in this process ANIMATION SHOWS THE PROCESS OF TRANSLOCATION PROKARYOTE PROTEIN SYNTHESIS In prokaryotes, the process is similar but there is no post transcriptional modification. The bacterial genes have no introns. Since prokaryotes have no nucleus, transcription and translation can occur at the same time as shown in the diagrams. Ribosomes are smaller in prokaryotes. They are 70s. PROKARYOTE PROTEIN SYNTHESIS cont’d When the stop signal is encountered, the ribosome disengages from the mRNA. The ribosomes of prokaryotes have a 30s subunit and a 50s subunit. Overall they are called 70s (sorry they don’t add up) 50s 30s POLYRIBOSOMES – several ribosomes copy the mRNA at the same time. Now the primary structure for the polypeptide is formed, the next phase involves chaperone proteins that assist the proteins in the folding process to form the proper shape. They can prevent proteins from denaturing in the folding process. A chaperone protein ? What is this? Some 1950’s prom? Well that pretty much explains the whole process of protein synthesis. We’ll bill ya. TRANSCRIPTION-TRANSLATION PROTEIN SYNTHESIS ENDS