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Chapter 17 From Gene to Protein Main Questions: The information content in DNA is the specific sequence of nucleotides along the DNA strands. How does this information determine the organism’s appearance? How is the information in the DNA sequence translated by a cell into a specific trait? Before Transcription and Translation Before these had been discovered, there was many questions as to what caused many metabolic errors. In 1909, Archibald Garrod was the first man to suggest that genes code for enzymes that dictate phenotypes of organisms. He called these “inborn errors of metabolism.” He used alkaptonuria as a example. George Beadle and Boris Ephrussi Proposed a similar hypothesis regarding eye pigment production in fruit flies. As time went on, biochemists performed numerous experiments that supported the hypotheses of Garrod, Beadle and Ephrussi. Experiments Demonstrating the Relationship Between Genes and Enzymes George Beadle and Edward Tatum experimented with bread mold (N. crassa) Bread mold is hearty. It can grow on minimal medium because it can use various pathways to synthesize everything it needs to survive. Minimal medium is culture medium that contains everything necessary for growth of the wild type: usually inorganic salts, a carbon source including vitamins and water. Beadle and Tatum Exposed the bread mold to X-rays to create a variety of mutants. Most mutants could survive on complete growth medium--medium supplied with vitamins and minerals. Looked to see how they responded to various growth medium--those lacking various nutrients required for growth. Looked closely at the pathway that synthesized arginine. Beadle and Tatum Found that each mutant was unable to carry out one step in the pathway for synthesizing arginine because it lacked the necessary enzyme. They also noticed that the mutants would survive if they were grown on a medium supplied with a compound made after the defective step. Beadle and Tatum’s Famous Experiment They hypothesized the arginine synthesis pathway. Examined 4 strains of N. crassa. Found mutants could only grow when the compound is supplied after the defect. Results confirmed their one gene-one enzyme hypothesis. Beadle and Tatum Somehow the gene that converts one product to the next in the pathway must have been affected by the X-rays. One gene must be responsible for one enzyme--their original hypothesis. One-Gene One-Enzyme Hypothesis As more was learned about proteins, it was determined that not all proteins are enzymes--(they are gene products, though) The hypothesis was revised: It is now the one gene-one polypeptide hypothesis. Many enzymes are comprised of multiple proteins. Even this is wrong because some genes code for RNA (that may not become protein). The Bridge Between DNA and Protein RNA is the single stranded compound that carries the message from the DNA to the ribosome for translation into protein. Recall, DNA = A,T,C,G; RNA= A,U,C,G The order of these bases carries the code for the protein which is constructed from any or all of the 20 amino acids. Transcription and Translation Going from gene to protein. Transcription is the synthesis of mRNA using DNA as the template. Similar to DNA synthesis. mRNA is the message (hence the “m”) from the gene. Translation is the process that occurs when the mRNA reaches the ribosome and protein synthesis occurs. RNA RNA is used because it is a way to protect the DNA from possible damage. Many copies of RNA can be made from one gene, thus, it allows many copies of a protein to be made simultaneously. Additionally, each RNA transcript can be translated repeatedly--via polyribosome. Polyribosome Here you can see an mRNA transcript being translated into many copies of protein by multiple ribosomes in a eukaryote. This is a way in which the cell can efficiently make numerous copies of protein. Polyribosome Here it is again in a prokaryote. The process essentially the same between prokaryotes and eukaryotes. One Main Difference Between prokaryotes and eukaryotes, there is one main difference between transcription and translation. The two processes can occur simultaneously in prokaryotes because they lack a nucleus. In eukaryotes, the two processes occur at different times. Transcription occurs in the nucleus, translation occurs in the cytoplasm. The Genetic Code Scientists began wondering how the genetic information contained within DNA instructed the formation of proteins. How could 4 different base pairs code for 20 different amino acids? 1:1 obviously didn’t work; a 2 letter code didn’t work either; but a 3 letter code would give you more than enough needed. The Genetic Code 61 of the 64 codons code for amino acids. 3 of the codons code for stop codons and signal an end to translation. AUG--start codon Genetic Code The genetic code is said to be redundant. More than one triplet codes for the same amino acid. One triplet only codes for one amino acid. The reading frame is important because any error in the reading frame codes for gibberish. Transcription The gene determines the sequence of bases along the length of the mRNA molecule. One of the two regions of the DNA serves as the template. The DNA is read 3’-->5’ so the mRNA can be synthesized 5’-->3’ Translation mRNA triplets are called codons. Codons are written 5’-->3’ Codons are read 5’-->3’ along the mRNA and the appropriate aa is incorporated into the protein according to the codon on the mRNA molecule. As this is done, the protein begins to take shape. mRNA and RNA Polymerase mRNA is the “messenger” or vehicle that carries the genetic information from the DNA to the protein synthesizing machinery. RNA polymerase pries apart the DNA and joins RNA nucleotides together in the 5’-->3’ direction (adding, again, to the free 3’ end). RNA polymerase is just like DNA polymerase, but it doesn’t need a primer. The Synthesis of mRNA RNA pol II encounters a promoter on the DNA near a transcriptional unit and starts synthesizing RNA. When the RNA pol II encounters a terminator sequence, transcription stops. The Synthesis of mRNA RNA pol II encounters a promoter on the DNA near a transcriptional unit and starts synthesizing RNA. When the RNA pol II encounters a terminator sequence, transcription stops. Different Types of RNA Polymerase Prokaryotes have one type of RNA polymerase that synthesizes mRNA and the other types of RNA as well. Eukarytoes have 3 different types in their nuclei (I, II, III). mRNA synthesis uses RNA pol II. Promoters Promoters are found on the DNA molecule and initiates the transcription of the gene. This is the site where transcription factors and RNA polymerase attach. Transcription is finished when the RNA polymerase reaches the terminator. The stretch of DNA that is transcribed is known as the transcription unit. Promoters Usually extend a few dozen nucleotides upstream from the transcription start point. Include a TATA box in eukaryotes. Promoters are important for the binding of the RNA polymerase. The Initiation of Transcription Transcription factors bind to the promoter region enabling RNA pol II to do so. The RNA pol II binds with additional transcriptional factors creating a transcription initiation complex. DNA unwinds and transcription begins. RNA pol II Promoter Differences Between Prokaryotes and Eukaryotes In prokaryotes, RNA polymerase recognizes and binds to the promoter on the DNA and immediately begins synthesizing mRNA. In eukaryotes, a group of proteins called transcription factors are needed for the binding of the RNA polymerase and the initiation of transcription. Promoter Differences Between Prokaryotes and Eukaryotes Once the transcription factors bind to the promoter, RNA pol II binds and transcription can then proceed. The entire group of proteins in the eukaryote are called the transcription initiation complex. Transcription As the RNA pol II moves along the DNA, it uncoils it, synthesizes the mRNA transcript and peels away from the DNA allowing it to recoil. Numerous RNA polymerases can transcribe the same DNA segment (protein) at the same time. This enables the cell to make large amounts of protein in a short period of time. Transcription An electron micrograph showing the transcription of 2 genes. Transcription Termination In prokaryotes transcription proceeds through a DNA sequence that functions as a termination signal causing the polymerase to detach from the DNA. This release of the transcript makes it immediately available for use as mRNA. Transcription Termination In eukaryotes, when the RNA pol II reads a certain signal sequence, it cleaves off the RNA from the growing chain as RNA pol II continues transcribing DNA. The RNA pol II continues to read and transcribe DNA and eventually falls off the DNA template strand, (not fully understood). The RNA produced now is still not ready for use. RNA Modification The eukaryotic RNA transcript now gets modified before it enters the cytoplasm. The 5’ end of the transcript gets modified before leaving the nucleus--a 5’ cap of nucleotides. The 3’ end is also modified--numerous adenine nucleotides--called a poly-A tail. Important Functions of the 5’ Cap and Poly-A Tail They facilitate export of the mature mRNA from the nucleus. They protect mRNA from degradation by hydrolytic enzymes. They assist in the attachment of the ribosome to the 5’ end of the mRNA. mRNA Modification The mRNA is further processed after the ends have been modified--RNA splicing. The initial transcript (~8000 bp) is reduced (to ~1200 on average). The large, non-encoding regions of the DNA that get transcribed are spliced out. Introns--intervening regions are removed. Exons--expressed regions are kept. mRNA Modification Some untranslated regions of the exons are saved because they have important functions such as ribosome binding. RNA splicing occurs via snRNP’s. snRPs consist of RNA and protein and join together to form a spliceosome which interacts with the intron to clip it out and join the exons together. So, Why is RNA Splicing Significant? In many genes, different exons encode separate domains of the protein product. RNA Splicing The way the RNA is spliced determines which proteins will be expressed. The different sexes of some organisms splice RNA differently and thus translate the genes into proteins differently--contributing to differences seen among sexes. The alternative RNA splicing is one possible reason humans can get by with relatively few genes. Translation Translation is when the cell interprets the genetic message and builds the polypeptide. tRNA acts as the interpreter. tRNA transfers aa’s from the cytoplasm to the ribosome where they are added to the growing polypeptide. All tRNA molecules are different. tRNA Structure and Function tRNA , like mRNA, is made in the nucleus and is used over and over again. tRNA has an aa at one end and an anticodon at the other end. The anticodon acts to base pair with the complementary code on the mRNA molecule. tRNA Structure and Function As tRNA reads the mRNA transcript, it brings an aa to the ribosome and adds it to growing polypeptide. The 2D shape is similar to a cloverleaf. tRNA tRNA is about 80 nucleotides long and full of complementary stretches of bases that H-bond to one another giving it a 3D shape like an “L.” tRNA From this, the anticodon loop comes out of one end and at the other end protrudes the 3’ end that carries the aa. 2 Recognition Steps in Translation 1. There must be a correct match between tRNA and an aa. 2. The accurate translation of the mRNA molecule. 1. The Correct Match 1. Each aa gets joined to a tRNA by aminoacyltRNA synthase--there are 20 of these, one for each amino acid. This enzyme catalyzes the attachment of aa to tRNA. The activated aa is now ready to deliver the aa to the growing polypeptide. 2. Accurate Translation The tRNA must correctly match up the tRNA anticodon with an mRNA codon. There is not a 1:1 ratio of the tRNA molecules with mRNA codons. Some tRNA’s can bind to more than one codon. This versatility is called “wobble.” 2. Accurate Translation Wobble enables tRNA to bind differently in one of its base pairs. This is why codons for some aa’s differ in their 3rd base. For example: the uracil at the 5’ end of a tRNA anticodon can pair with an A or a G in the third position of the 3’ end of the mRNA codon. Ribosomes These are the sites of protein synthesis. They consist of a large and a small subunit and are comprised of RNA and protein. The RNA is ribosomal RNA (rRNA). Bacterial (70s, 50S + 30s) Eukaryotic (80s, 60S + 40s) Ribosomes rRNA genes are found on chromosomal DNA and are transcribed and processed in the nucleolus. They are assembled and transferred to the cytoplasm as individual subunits. The large and small subunits form one large subunit when they are attached to the mRNA. Ribosomes The structure of ribosomes fit their function. They have an mRNA binding site, a P-site, an A-site and an E-site. P-site (peptidyl-tRNA) holds the tRNA carrying the growing peptide chain. A-site (aminnoacyl-tRNA) holds the tRNA carrying the next aa to be added to the chain. E-site is the exit site where the tRNAs leave the ribosome. Each of these are binding sites for the mRNA. QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see this picture. rRNA rRNA is the most abundant form of RNA in the cell because there are thousands of ribosomes within a cell and about 2/3’s the mass of a ribosome is rRNA. The 3 Stages of Protein Building 1. Initiation 2. Elongation 3. Termination All three stages require factors to help them “go” and GTP to power them. 1. Initiation Initiation brings together mRNA, tRNA and the 2 ribosomal subunits. Initiation factors are required for these things to come together. GTP is the energy source that brings the initiation complex together. 2. Elongation The elongation stage is where aa’s are added one by one to the growing polypeptide chain. Elongation factors are involved in the addition of the aa’s. GTP energy is also spent in this stage. 3. Termination Termination occurs when a stop codon on the mRNA reaches the “A-site” within the ribosome. Release factor then binds to the stop codon in the “A-site” causing the addition of water to the peptide instead of an aa. This signals the end of translation. Polypeptide Synthesis As the polypeptide is being synthesized, it usually folds and takes on its 3D structure. Post-translational modifications are often required to make the protein function. Adding fats, sugars, phosphate groups, etc.. Removal of certain proteins to make the protein functional. Separately synthesized polypeptides may need to come together to form a functional protein. Eukaryotic Ribosomes Recall the 2 types: Free and bound. They function exactly the same and can switch from free to bound. This switch can occur when the protein that is being translated contains a signal peptide instructing the ribosome to attach to the ER. Once attached to the ER, synthesis will continue to completion and can then be exported from the cell. Signal Peptide Recognition The signal peptide is recognized as it emerges from the ribosome by a protein-RNA complex called signal-recognition particle. The particle functions by bringing the ribosome to a receptor protein built into the ER where synthesis continues and the growing peptide finds its way into the lumen. Signal Peptide Recognition Once in the lumen of the ER, the newly synthesized polypeptide is modified. The signal peptide is cut out by an enzyme. The protein then undergoes further processing and is shipped where it needs to go. 3 Useful Properties of RNA 1. It can H-bond to other nucleic acids. 2. It can form a specific 3D shape by Hbonding on itself. 3. It has functional groups that allow it to act as a catalyst. Differences in Prokaryotic and Eukaryotic Gene Expression Prokaryotic and eukaryotic RNA polymerases are different, but perform the same function. Transcription is terminated differently. Prokaryotic and eukaryotic ribosomes are different. Transcription and translation are streamlined in prokaryotes, it is compartmentalized in eukaryotes. Eukaryotic cells have a complex system of targeting proteins for their final destination. Mutations Mutations are changes in genetic material. They can cover large portions of DNA or they can be a point mutation. There are 2 broad categories of point mutations: Base-pair substitutions Insertions or deletions Mutations can be caused by a number of things: Errors in replication, recombination, or repair Mutagens--X, , UV radiation, chemicals Spontaneous Base-Pair Substitutions One base pair gets substituted for another. Sometimes these are silent because of the redundancy of the genetic code. Other times they are silent because the mutation codes for an aa/protein that is not essential to the protein’s function. Also, there are times when the two aa’s behave the same even though they are different. Missense Mutations Vs. Nonsense Mutations Missense mutations code for an aa, usually the wrong aa. In a nonsense mutation, the wrong aa is encoded and it usually results in a stop codon and the production of a nonfunctional protein. Insertions and Deletions These are often referred to as “frame-shift” mutations because they alter the reading frame of the mRNA. They almost always result in a nonfunctional protein.