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Auburn University
BIOL 1020
Anne- Marie Singh

BIOL 1020 – CHAPTER 17 LECTURE NOTES Chapter 17: From gene to protein I. Genes generally are information for making specific protein a. in connection with the rediscovery of Mendel’s work around the dawn of the 20th century, the idea that genes are responsible for making enzymes was advanced b. this view was summarized in the classic work Inborn Errors of Metabolism (Garrod 1908) c. work by Beadle and Tatum in the 1940s refined this concept i. found mutant genes in the fungus Neurospora that each affected a single step in a metabolic pathway ii. developed the “one gene, one enzyme” hypothesis 1. later work by Pauling and others showed that other proteins are also generated genetically 2. also, some proteins have multiple subunits encoded by different genes 3. this ultimately led to the “one gene, one polypeptide” hypothesis II. RNA (ribonucleic acid) a. RNAserves mainly as an intermediary between the information in DNAand the realization of that information in proteins b. RNAhas some structural distinctions from DNA i. typically single-stranded (although often with folds and complex 3° structure) ii. sugar is ribose; thus, RNApolymers are built from ribonucleotides iii. uracil (U) functions in place of T c. three main forms of RNAare used: mRNA, tRNA, and rRNA i. mRNA or messenger RNA: copies the actual instructions from the gene ii. tRNA or transfer RNA: links with amino acids and bring them to the appropriate sites for incorporation in proteins iii. rRNA or ribosomal RNA: main structural and catalytic components of ribosomes, where proteins are actually produced iv. all are synthesized from DNAtemplates (thus, some genes code for tRNAand rRNA, not protein) III. Overview of gene expression a. Central Dogma of Gene Expression: DNARNAprotein i. the gene is the DNAsequence with instructions for making a product ii. the protein (or protein subunit) is the product b. DNA RNAis transcription i. making RNAusing directions from a DNAtemplate ii. transcribe = copy in the same language (language used here is base sequence) c. RNA protein is translation i. making a polypeptide chain using directions in mRNA ii. translate = copy into a different language; here the translation is from base sequence to amino acid sequence d. there are exceptions to the central dogma i. some genes are for an RNAfinal product, such as tRNAand rRNA(note: mRNAis NOT considered a final product) ii. for some viruses use RNAas their genetic material 1. some never use DNA 2. some use the enzyme reverse transcriptase to perform RNADNAbefore then following the central dogma IV. Transcription: making RNA from a DNA template a. RNAis synthesized as a complementary strand using DNA- dependent RNA polymerases i. process is somewhat similar to DNAsynthesis, but no primer is needed ii. bacterial cells each only have one type of RNA polymerase iii. eukaryotic cells have three major types of RNA polymerase 1. RNApolymerase I is used in making rRNA 2. RNApolymerase II is used in making mRNAand some small RNAmolecules 3. RNApolymerase III is used in making tRNAand some small RNAmolecules b. only one strand is transcribed, with RNApolymerase using ribonucleotide triphosphates (rNTPs, or just NTPs) to build a strand in the 5’ 3’ direction i. thus, the DNAis transcribed (copied or read) in the 3’5’ direction ii. the DNAstrand that is read is called the template strand or sense strand iii. upstream means toward the 5’end of the RNAstrand, or toward the 3’end of the template strand (away from the direction of synthesis) iv. downstream means toward the 3’end of the RNAstrand, or toward the 5’end of the template strand c. transcription has three stages: initiation, elongation, and termination d. initiation requires a promoter – site where RNApolymerase initially binds to DNA i. promoters are important because they are needed to allow RNAsynthesis to begin ii. promoter sequence is upstream of where RNAstrand production actually begins iii. promoters vary between genes; this is the main means for controlling which genes are transcribed at a given time iv. bacterial promoters 1. about 40 nucleotides long 2. positioned just before the point where transcription begins 3. recognized directly by RNApolymerase v. eukaryotic promoters (for genes that use RNA polymerase II) 1. initially, transcription factors bind to the promoter; these proteins facilitate binding of RNA polymerase to the site 2. transcription initiation complex a. completed assembly of transcription factors and RNApolymerase at the promoter region 3. allows initiation of transcription (the actual production of an RNAstrand complementary to the DNAtemplate) 4. genes that use RNApolymerase II commonly have a “TATAbox” about 25 nucleotides upstream of the point where transcription begins a. actual sequence is something similar to TATAAAon the non-template strand b. sequences are usually written in the 5’3’ direction of the strand with that sequence unless noted otherwise vi. regardless of promoter specifics, initiation begins when RNApolymerase is associated with the DNA 1. RNApolymerase opens and unwinds the DNA 2. RNApolymerase begins building an RNAstrand in the 5’3’direction, complementary to the template strand 3. only one RNAstrand is produced e. elongation i. RNApolymerase continues building the RNAstrand in a linear fashion, unwinding and opening up the DNAalong the way ii. the newly synthesized RNAstrand easily separates from the DNAand the DNAmolecule “zips up” behind RNA polymerase, reforming the double helix f. termination: the end of RNAtranscription i. in prokaryotes, transcription continues until a terminator sequence is transcribed that causes RNApolymerase to release the RNAstrand and release from the DNA ii. termination in eukaryotes is more complicated and differs for different RNApolymerases 1. still always requires some specific sequence to be transcribed 2. for RNApol II the specific sequence is usually hundreds of bases before the actual ending site V. The genetic code a. the actual information for making proteins is called the genetic code b. the genetic code is based on codons: sequences of three bases that instruct for the addition of a particular amino acid (or a stop) to a polypeptide chain i. codons are thus read in sequences of 3 bases on mRNA, sometimes called the triplet code ii. codons are always written in 5’3’fashion iii.four bases allow 43 = 64 combinations, plenty to code for the 20 amino acids typically used to build proteins iv. thus, a 3-base or triplet code is used v. see the genetic code figure 1. don’t try to memorize the complete genetic code 2. do know that the code is degenerate or redundant: some amino acids are coded for by more than one codon (some have only one, some as many as 6) 3. know that AUG is the “start” codon: all proteins will begin with methionine, coded by AUG 4. know about the stop codons that do not code for an amino acid but instead will end the protein chain 5. be able to use the table to “read” an mRNA sequence vi. the genetic code was worked out using artificial mRNAs of known sequence vii. the reading of the code 3 bases at a time establishes a reading frame; thus,AUG is very important as the first codon establishes the reading frame viii. the genetic code is nearly universal – all organisms use essentially the same genetic code (strong evidence for a common ancestry among all living organisms) c. mRNA coding region i. each mRNAstrand thus has a coding region within it that codes for protein synthesis ii. the coding region starts with theAUG start, and continues with the established reading frame iii. the coding region ends when a stop codon is reached iv. the mRNAstrand prior to the start codon is called the 5’untranslated region or leader sequence v. the mRNAstrand after the stop codon is called the 3’ untranslated region or trailing sequence vi. collectively, the leader sequence and trailing sequence are referred to as noncoding regions of the mRNA VI. Translation: using information in mRNA to direct protein synthesis a. in eukaryotes, mRNAis moved from the nucleus to the cytoplasm (in prokaryotes, there is no nucleus so translation can begin even while transcription is underway – see polyribosomes later) b. the site of translation is the ribosome i. ribosomes are complexes of RNAand protein, with two subunits ii. ribosomes catalyze translation (more on this role later) c. ultimately, peptide bonds must be created between amino acids to form a polypeptide chain i. recall that peptide bonds are between the amino group of one amino acid and the carboxyl group of another ii. primary polypeptide structure is determined by the sequence of codons in mRNA iii. the ribosome acts at the ribozyme that catalyzes peptide bond formation d. tRNAs bring amino acids to the site of translation i. tRNAs are synthesized at special tRNAgenes ii. tRNAmolecules are strands about 70-80 bases long that form complicated, folded 3-dimensional structures iii. tRNAs have attachment sites for amino acids iv. each tRNAhas an anticodon sequence region that will form a proper complementary basepairing with a codon on an mRNAmolecule v. tRNAis linked to the appropriate amino acid by enzymes called aminoacyl-tRNA synthetases 1. the carboxyl group of each specific amino acid is attached to either the 3' OH or 2' OH group of a specific tRNA 2. there is at least one specific aminoacyl-tRNA synthetase for each of the 20 amino acids used in proteins 3. ATP is used as an energy source for the reaction 4. the resulting complex is an aminoacyl-tRNA; this is also called a charged tRNA or activated tRNA 5. the amino acid added must be the proper one for the anticodon on the tRNA vi. there are not actually 64 different tRNAs 1. three stops have no tRNA 2. some tRNAs are able to be used for more than one codon a. for these, the third base allows some “wobble” where basepairing rules aren’t strictly followed b. this accounts for some of the degeneracy in the genetic code c. for note how often the 3rd letter in the codon does not matter in the genetic code d. there are usually only about 45 tRNAtypes made by most organisms e. the mRNAand aminoacyl-tRNAs bond at the ribosome for protein synthesis i. the large ribosome subunit has a groove where the small subunit fits ii. mRNAis threaded through the groove iii. the large ribosomal subunit has two depressions where tRNAs attach (Aand Pbinding sites), and a third site called the exit site (E site) 1. the E site is where uncharged tRNAmolecules are moved and then released 2. the Psite is where the completed part of the polypeptide chain will be attached to tRNA 3. the Asite is where the new amino acid will enter on an aminoacyl-tRNAas a polypeptide is made iv. the tRNAs that bond at these sites basepair with mRNA 1. pairing is anticodon to codon 2. must match to make proper basepairs, A-U or C-G, except for the allowed wobbles at the 3rd base f. translation has three stages: initiation, elongation, and termination i. all three stages have protein “factors” that aid the process ii. many events within the first two stages require energy, which is often supplied by GTP (working effectively like ATP) g. initiation – start of polypeptide production 1. an initiation complex is formed 2. begins with the loading of a special initiator tRNA onto a small ribosomal subunit a. the initiator tRNArecognizes the codon AUG, which is the initiation start codon b. AUG codon codes for the amino acid methionine c. the initiator tRNAthus is charged with methionine; written as tRNAMet 3. next the small ribosomal subunit binds to
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