Introduction to Molecular Genetics (http://chemwiki.ucdavis.edu/wikitexts/sacramento_city_college/scc%3a_chem_309_(bennett)/chapters/17% 3A_ucleic_Acids) (http://www.nature.com/scitable/topicpage/ribosomes-transcription-and-translation-14120660) (http://www.nature.com/scitable/topicpage/translation-dna-to-mrna-to-protein-393) I. Introduction: A. Why you are like your parents, and your kids are (will be) like you? 1. Inherited traits result from the transfer of specific molecules to offspring. 2. All living things use DA to store inherited information and transfer it to offspring. 3. Some viruses use RA for information storage. B. rganisms organize their genetic information. 1. Chromosomes a. Contains one molecule of DA. b. umans have pairs in the nucleus of their cells. ne set from mom and one set from dad. Karyotype* of a human male. Courtesy: ational uman Genome Research Institute - Extracted image from http://www.genome.gov/glossary/resource s/karyotype.pdf.* The number and appearance of the chromosomes in the nucleus of a eukaryotic cell. If stretched out, the DA in chromosome 1 is roughly long. c. Chromosome pairs 1-22 are called autosomes and the 23 rd pair are chromosomes. XX indicates a female. XY indicates a male. d. ther organisms have different numbers of chromosomes. Bacteria have just one. 2. Genes A gene codes for one protein chain. 3. uman genome Complete set of DA that consists of: -the 23 pairs of chromosomes in the nucleus and -a small piece of DA in each mitochondria. Contains roughly genes. (< 2% of DA) 4. Expression of Genes a) ot all genes are expressed (their proteins made) all the time in all cells 1
b) Their expression is regulated. This allows for cells to be different, as a blood cell is different from a kidney cell. c) Also allows for cells to make different proteins only when they are needed. d) Some of the 98% of non-coding DA is involved in regulation of gene expression. C. The Central Dogma of molecular biology: DA makes RA makes Protein. Cell II. UCLEIC ACID CMPETS A. Polymers made from nucleotide monomers 1.Two main types a. RA: ribonucleic acid b. DA: 2'-deoxyribonucleic acid 2. A nucleotide has 3 components a. base b. sugar c. phosphate(s) - P - phosphate 2 base suga
B. itrogenous Bases (or just bases) 1. RA has mostly guanine (G), cytosine (C), adenine (A), & uracil (U) 2. DA has guanine (G), cytosine (C), adenine (A), & thymine (T) in place of U. ow do U and T differ? Identify hydrogen bond donor d & acceptor sites a. Show all the nonbonding pairs on the acceptor sites. ote: itrogen atoms with 3 single bonds cannot act as -bond acceptors. 3. ydrogen bonding sites are extremely important 4. Purines: G & A (two rings) Pyrimidines: C, T, & U. A G C T U R R is where the base attaches to the rest of the molecule. R R R R C. Sugar 1. Ribose in RA 2. 2'-Deoxyribose in DA ote: carbons are labeled with primes. ow do ribose and deoxyribose differ? 3
3. ucleoside: a base linked to sugar C 5' 2 4' 1' 3' 2' Adenosine D. Phosphate(s) 1. Usually linked to 5' C of the sugar 2. A nucleotide can have 1, 2 or 3 sugars to make mono-, di-, or triphosphates. E. ucleoside triphosphates are the monomers that make up RA and DA Example: ATP, adenosine triphosphate 1. Structure 2. ATP in metabolism. (The phosphodiester bond is a very high energy bond!) III. STRUCTURE F DA AD RA A. The first (1 o ) level is the sequence of nucleotides in the polymer chain 1. Like proteins, nucleic acid polymers have distinct ends: the 5'-end &. (see structure on next page.) 2. To find direction of chain, pick a sugar. Find 5'- & 3'- carbons, determine which end is which. 4
DA Sugar-phosphate Backbone - P - 5' base 1 3' P - 5' base 2 3' P - 5' base 3 3' B. Secondary structure: The Double elix!!!!!! 1. Determination of the double helical nature of DA was among the most important biology-chemistry achievements in the 20 th century. a) Circa 1950, people knew: i) DA had regular, repeating structures ii) A & T occur in = %, as do C & G b) James Watson & Francis Crick (W-C) used this info to propose a structural model for DA. c) The structure of this model immediately clarified the manner in which DA could be accurately copied. 2. Key to determining DA structure was seeing the importance of complimentary ydrogen Bonding between A & T and G & C. a) A always bonds with ; G always bonds with b) The sequence information is encoded in both strands. c) What does that indicate about making a new DA copy? 5
3. The two strands are oriented in opposite directions. This is called. a. Strands run (5' - 3') in opposite directions b. Strands align so bases can hydrogen bond. c. G always pairs with C (Three hydrogen bonds) d. A always pairs with T (Two hydrogen bonds) e. Sequences on the two strands are complimentary 4. Look at the DA double helix. Bacteriophage lambda operator DA (largely in B form) (This figure was prepared from pdb file 1lmb, which is the lambda repressor protein bound to its operator DA. The repressor functions to regulate transcription (see below) from nearby genes. The file was transferred to CACE and the protein deleted to aid in viewing.) 6
Doubled standed color emphasis Detail showing W-C base pairing View along helical axis for 1/2 turn https://en.wikipedia.org/wiki/introduction_to_genetics See narrow groove. C. Chromosomal structure: see: http://www.youtube.com/watch?v=luesmdr40 (http://www.nature.com/scitable/topicpage/eukaryotic-genome-complexity-437) 1. It runs from full folded, wrapped, etc. chromosome down to double helix. 2. Folded structure necessary to condense length of DA 3. Initial step of folding involves wrapping DA around a group of proteins (histones). IV. DA REPLICATI (initiate-elongate-terminate) A. DA replication is semi-conservative. 1. Parent DA strands separate (at specific sites). 2. Each parent strand serves as a template to make a new daughter strand. 7
3. This gives 2 half-new complementary strands. Emphasize: a) A pairs only with T b) G pairs only with C DA replication is semi-conservative. Parental DA molecule Two daughter DA molecules each contain one parental strand and one new strand. B. Many enzymes & proteins are involved in DA replication. 1. The main protein involved in making the new DA copies is called DA polymerase. (3 different types of polymerases!) 2. Some proteins help polymerase get started. 3. ther proteins help the DA unwind and keep short stretches of DA single-stranded. 4. Still other proteins help rewind & terminate. C. DA polymerases synthesize the new strand in the 5' to 3' direction. http://www.dnai.org/a/index.html go to Copying the Code or http://www.youtube.com/watch?v=bee6pwugpo8 1. Leading strand: topologically 5' to 3' direction. 2. Lagging strand: looks 3' 5' direction, but is actually 5' 3' in short (100 base pair) chunks. D. Many bacterial DA s are circular. E. Is your nuclear DA circular or linear? 1. Specific structure at end called telomere. Contains many repeating 6 nucleotide sequences. 2. Synthesis of linear DA leads to problems with shortened ends. Telomerase, a specialized enzyme, can add repeats. This activity is low in human cells. So eventually cell division ends and cell death is initiated. May play a role in aging. 3. Cancer cells have enhanced telomerase activity. Telomerase might be a possible target for anti-cancer drugs. V. IFRMATI FLW I BILGICAL SYSTEMS DA RA protein A. Main types of RA: 1. Messenger RA (mra) 2. Transfer RA (tra) 3. Ribosomal RA (rra) 4. ther RAs 8
B. mra codes for the synthesis of proteins. The largest part of your DA that codes for RA codes for this type 1. Bacterial mras correlate directly with the genes that code for them. 2. Most of your mras are made from much larger precursors that you trim down to the right size. a) Eucaryotic genes often contain introns. Introns (or intervening sequences) are spliced out of the initial RA molecule shortly after it is made. b) We make additional chemical modifications to the 5' and 3' ends of the mras before they are used in protein synthesis. C. tra serves an important translational function in protein synthesis at the ribosome. 1. At the ribosome one end (anti-codon loop) of tras binds (by W-C base pairing) to complimentary RA triplets in the mra. 2. The other end covalently links to an amino acid. 3. The middle provides recognition sites so the tra charging enzymes will link the correct amino acid to the correct tra. By Yikrazuul - wn work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=10126790 By Yikrazuul - wn work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=1031209 7 D. rra is an important catalytic and structural component of the ribosomes. Ribosomes contain more than one rra. E. ther RAs. There are many, but they are slightly beyond our scope. 9
F. Transcription is catalyzed by RA polymerase. See dnai.org Copying the Code. Again, the pattern is: 1. Initiation (at specific sites identified by specific DA sequences) 2. Elongation (70- a few thousand bases are added) 3. Termination (at specific sites identified by specific DA sequences) Initiation of transcription By Forluvoft - wn work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=2892141 Transcription elongation By Forluvoft - wn work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=2892073 G. Many eukaryotic mras are processed before being translated into the amino acid sequences of proteins. 1. Most of your mras are made from much larger precursors that you trim down to the right size. 2. Eukaryotic genes often contain noncoding sequences (called intervening sequences or introns), that are removed or are spliced out of the initial RA molecule shortly after it is made 3. Coding regions (exons) are spliced together to form a continuous strand. 4. The ends are protected. The 5' end is capped, & the 3' end has poly A added. 5. Some mras: intron RA is 10-30 times longer than exon RA. 6. Exons make up 1.5% of our DA. VI. TE GEETIC CDE (You don t need to memorize it.) A. ow do you get from the language of nucleotides to the language of amino acids? 1. ow many aa s could you code for using a 4 base alphabet (A, U, G, & C) and one letter long words? 2. ow many aa s could you code for using a 4 base alphabet and two letter long words? 3. ow many aa s could you code for using a 4 base alphabet and three letter long words? 4. ow many common amino acids are there? 10
The words are called triplet codons. mra Codons: The Genetic Code U C A G UUU Phe UCU Ser UAU Tyr UGU Cys U U UUC Phe UCC Ser UAC Tyr UGC Cys C UUA Leu UCA Ser UAA Stop UGA Stop A UUG Leu UCG Ser UAG Stop UGG Trp G CUU Leu CCU Pro CAU is CGU Arg U C CUC Leu CCC Pro CAC is CGC Arg C CUA Leu CCA Pro CAA Gln CGA Arg A CUG Leu CCG Pro CAG Gln CGG Arg G AUU Ile ACU Thr AAU Asn AGU Ser U A AUC Ile ACC Thr AAC Asn AGC Ser C AUA Ile ACA Thr AAA Lys AGA Arg A AUG met ACG Thr AAG Lys AGG Arg G GUU Val GCU Ala GAU Asp GGU Gly U G GUC Val GCC Ala GAC Asp GGC Gly C GUA Val GCA Ala GAA Glu GGA Gly A GUG Val GCG Ala GAG Glu GGG Gly G B. Comments on genetic code: See for example: 1. It is redundant, multiple codons for most aa. 2. Three of the codons are stop signals. 3. The code is nearly universal. (re. evolution) Bacteria use the same code we do. C. Codons are within the mra. An anti-codon is located in the anticodon loop of each tra. By Yikrazuul - wn work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid =10312097 11
VII. PRTEI SYTESIS A. Energetics of protein synthesis. ΔG = Δ - TΔS 1. Is this rxn. favored: protein + 2 amino acids What does meat tenderizer do, and how does it work? 2. Since G of free amino acids is too low for their use as reactants in protein synthesis, we need to make a higher G reactant. (Graph) 3. Amino acids are activated (converted to higher G compounds): a) by reaction with ATP followed by b) ester linkage to 3' ribose of proper tra c) rxns. a) & b) catalyzed by aminoacyl tra synthetases. There is essentially a different charging enzyme for each different tra. See dnai.org Reading the Code. B. Three major stages: 1. initiation 2. elongation 3. termination Aside on ribosomes (from rat liver unless noted): a) They are giant enzymes. (MW = 4.22 x 10 6 ) What rxn. do they catalyze? b) 2 subunits 40S/60S (30S/50S in prokaryotes) c) 82 different proteins (40% of weight) d) 4 different rras (60% of weight) See pdb structures 1ffk and 1gix. By Boumphreyfr - wn work, CC BY- SA 3.0, https://commons.wiki media.org/w/index.ph p?curid=7200200 12
C. Initiation 1. mra, small ribosomal subunit, & charged initiator tra met form ternary complex. A specific region (base sequence) of the mra binds the ribosomal subunit. What minimal 3 base sequence of the mra must be involved? 2. A number of initiation factors (protein) also act. 3. Large ribosomal subunit now binds. 4. Aside on ribosomes (from rat liver unless noted): a) They are giant enzymes. (MW = 4.22 x 10 6 ) What rxn. do they catalyze? b) 2 subunits 40S/60S (30S/50S in prokaryotes) c) 82 different proteins (40% of weight) d) 4 different rras (60% of weight) See pdb structures 1ffk and 1gix. 5. GTP hydrolysis is required. (Why do we need to take in energy?) D. Elongation 1. ext charged tra xxx binds to the complex at the aminoacyl (A) site of the ribosome. XXX determined by anti-codon:codon base pairing. 2. C-terminal end of growing peptide chain forms peptide bond with amino group of aa XXX bound to tra at A site. 3. Empty tra dissociates from P site. 4. Peptidyl-tRA at A site is translocated to P site. Requires GTP. 5. Elongation factors (protein) are involved. E. Termination 1. When a stop codon is encountered, termination occurs. 2. Protein release factors (not tra) recognize stop codons. 3. Specific sequences often occur near stop codon that aid in termination. F. General protein synthesis comments. 1. mra read 5' 3' 2. Protein synthesized from - to C-terminal end. 3. More than one ribosome can read an mra at a time. (Amplification!) 4. Very few errors occur. VIII. MUTATIS, DA REPAIR & GEETIC DISEASES A. Mutations 1. A mutation is a change (fixed) in DA. 2. Point mutations (a base change) a) Silent ex: DA change resulting in mra codon change of UUU (phe) UUC (phe) 13
b) Missense Conservative ex: mra codon change of AUU (Ile) GUU (val) ot conservative Cause substantial change in protein function. For sickle-cell anemia, can you figure out the DA change from Glu Val? c) onsense Change of an aa-determining codon to a stop codon. Results in early termination of protein synthesis 3. Frame Shift: Result of addition or deletion. AUG UCG AAU CAC AGA met - ser - asn - his - arg AUG UUC GAA UCA CAG A met - phe - glu - ser - gln 4. Deletion (can be of one base or 1000 or more) B. Mutagens 1. Mutagens cause mutations 2. We think of mutations as random, but specific mutagens do not necessarily occur or act randomly. 3. UV light can cause mutations (Thymine dimers). 4. Why does DA have T, not U? You correct 1 million of these/day. Could you correct these if your DA normally contained U? 5. Mutagens maybe also be carcinogens. C. Examples of genetic diseases occur when a mutation has a deleterious effect. 1. Sickle-cell anemia 2. Xeroderma pigmentosum (thymine dimer repair) 14