Introduction to Molecular Genetics

Transcription

1 Introduction to Molecular Genetics I. Introduction: A. ave you thought much about 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. ome viruses use A for information storage. B. rganisms organize their genetic information. 1. Chromosomes a. umans have 23 pairs. b. Bacteria may have just one. 2. Genes 3. Many ways genes are organized and their expression is controlled. C. The Central Dogma of molecular biology: DA makes A makes rotein. transcription translation DA A rotein replication reverse transcription Cell II. UCLEIC ACID CMET A. Two main types of nucleic acids 1. A: ribonucleic acid 2. DA: 2'-deoxyribonucleic acid 3. Long, linear chains made from small units or nucleotides 1

2 B. A nucleotide has 3 components 1. base 2. sugar 3. phosphate(s) - base - phosphate sugar C. itrogenous Bases 1. A 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. A G T U 2 C Use "d" for the donor sites. Draw non-bonding pairs for acceptor sites: ow do U and T differ? a) Bases have hydrogen bonding sites that are extremely important b) Identify all sites in & on 6-membered rings above 3. urines: G & A (two rings) yrimidines: C, T, & U. A G C T U is where the base attaches to the rest of the molecule. 2

3 C. ugar 1. ibose in A 2. 2'-Deoxyribose in DA 3. ote: carbons are labeled with primes. 4. ow do ribose and deoxyribose differ? 5. (A nucleoside is a base linked to sugar) 5' C 2 4' 3' C 2 2' ribosyl BAE 1' BAE D. hosphate(s) 1. Usually linked to 5' C of the sugar 2'-deoxyribosyl 2. A nucleotide can have 1, 2 or 3 sugars to make mono-, di-, or triphosphates. hosphate, mono-, di- and tri AT, adenosine triphosphate, is an important nucleotide in metabolism. 2 AT structure III. TUCTUE F DA AD A 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 & 3'-end. (see structure on next page.) 2. To find direction of chain, pick a sugar. Find 5'- & 3'- carbons, determine which end is which. 3

4 DA ugar-phosphate Backbone - - 5' base 1 3' - 5' base 2 3' - 5' base 3 3' B. DA is double-stranded and helical 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) Watson & Crick (W-C) used this info to propose a structural model for DA. c) The structure of this model immediately clarified many aspects of DA function. 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 T; G always bonds with C b) The sequence information is encoded in both strands. c) What does that indicate about making a new DA copy? A T G C 4

5 3. The two strands are oriented in opposite directions ( anti-parallel ) a. trands run (5' - 3') in opposite directions b. trands align so bases can hydrogen bond. 5' G C 3' c. G always pairs with C (Three hydrogen bonds) d. A always pairs with T (Two hydrogen bonds) A T e. equences on the two strands are complimentary C G T A T A 3' 5' 4. Look at at DA double helix. (ee next page.) (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.) 5

6 Bacteriophage lambda operator DA (largely in B form) 6

7 Doubled standed color emphasis Detail showing W-C base pairing View along helical axis for 1/2 turn 7

8 C. Chromosomal structure see: ( 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 ELICATI (initiate-elongate-terminate) Go to dnai.org & click on CDE. Go to Finding the structure, then to problem, pieces of the puzzle, and putting it together. (Do the interactives!!!) A. DA replication is semi-conservative. 1. arent DA strands separate (at specific sites). 2. Each parent strand serves as a template to make a new daughter strand. 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. arental 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. ome proteins help polymerase get started. 3. ther proteins help the DA unwind and keep short stretches of DA single-stranded. 4. till other proteins help rewind & terminate. C. DA polymerases synthesize the new strand in the 5' to 3' direction. Therefore: at dnai.org go to Copying the Code 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. 8

9 E. Is your nuclear DA circular? 1. pecific structure at end called telomere. Contains many repeating 6 nucleotide sequences. 2. ynthesis of linear DA leads to problems with shortened ends. Telomerase, a specialized enzyme, can add repeats. This activity is low in human cells. o eventually cell division ends and cell death is initiated. May play a role in aging. 3. ome immortal (including cancer) cells have enhanced telomerase activity. V. IFMATI FLW I BILGICAL YTEM DA A protein A. Main types of A: 1. Messenger A (ma) 2. Transfer A (ta) 3. ibosomal A (ra) 4. ther As B. ma codes for the synthesis of proteins. The largest part of your DA that codes for A codes for this type (how many different mas! ) 1. Bacterial mas correlate directly with the genes that code for them. 2. Most of your mas are made from much larger precursors that you trim down to the right size. Draw picture on the board. a) Eucaryotic genes often contain introns. Introns (or intervening sequences) are spliced out of the initial A molecule shortly after it is made. b) We make additional chemical modifications to the 5' and 3' ends of the mas before they are used in protein synthesis. C. ta serves an important translational function in protein synthesis at the ribosome. 1. At the ribosome one end (anti-codon loop) of tas binds (by W-C base pairing) to complimentary A triplets in the ma. 2. The other end covalently links to an amino acid. 3. The middle provides recognition sites so the ta charging enzymes will link the correct amino acid to the correct ta. D. ra is an important catalytic and structural component of the ribosomes. ibosomes contain more than one ra. E. ther As. There are many, but they are slightly beyond our scope. 9

10 F. Transcription is catalyzed by A polymerase. ee 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) G. Many eukaryotic mas are processed before being translated into the amino acid sequences of proteins. 1. oncoding sequences, called introns, are removed. 2. Coding regions (exons) are spliced together to form a continuous strand, the 5' end is capped, & the 3' end has poly A added. 3. In some mas: intron A is times longer than exon A. 4. 3% of our DA appears to code for proteins. 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? The words are called triplet codons. B. Comments on genetic code: ee 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) a) Bacteria use the same code we do. b) Exceptions to pattern fit nicely into established evolutionary patterns. i) mostly mitochondrial ( proteins) ii) derivation from an original code 10

11 ma Codons: The Genetic Code U C A G UUU he UCU er UAU Tyr UGU Cys U U UUC he UCC er UAC Tyr UGC Cys C UUA Leu UCA er UAA top UGA top A UUG Leu UCG er UAG top UGG Trp G CUU Leu CCU ro CAU is CGU Arg U C CUC Leu CCC ro CAC is CGC Arg C CUA Leu CCA ro CAA Gln CGA Arg A CUG Leu CCG ro CAG Gln CGG Arg G AUU Ile ACU Thr AAU Asn AGU er U A AUC Ile ACC Thr AAC Asn AGC er 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 C. Codons are within the ma. An anti-codon is located in the anticodon loop of each ta. ee pdb structure 4tna. VII. TEI YTEI A. Energetics of protein synthesis. G = - T 1. Is this rxn. favored: protein + 2 amino acids What does meat tenderizer do, and how does it work? 2. If rxn.: protein + 2 amino acids has negative G, can the reverse rxn. have negative G (be favored?)? 3. ince 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) 4. If proteins are less stable than their monomers, why don t we (our proteins) just fall apart? 11

12 5. Amino acids are activated (converted to higher G compounds): a) by reaction with AT followed by b) ester linkage to 3' ribose of proper ta c) rxns. a) & b) catalyzed by aminoacyl ta synthetases. Essentially a different charging enzyme for each different ta. ee animation. ee also at dnai.org eading the Code. B. Three major stages: 1. initiation 2. elongation 3. termination C. Initiation ( 1. ma, small ribosomal subunit, & charged initiator ta met form ternary complex. A specific region (base sequence) of the ma binds the ribosomal subunit. What minimal 3 base sequence of the ma 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 40/60 (30/50 in prokaryotes) c) 82 different proteins (40% of weight) d) 4 different ras (60% of weight) ee pdb structures 1ffk and 1gix. 5. GT hydrolysis is required. (Why do we need to take in energy?) D. Elongation 1. ext charged ta 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 ta at A site. 3. Empty ta dissociates from site. 4. eptidyl-ta at A site is translocated to site. equires GT. 5. Elongation factors (protein) are involved. 12

13 E. Termination 1. When a stop codon is encountered, termination occurs. 2. rotein release factors (not ta) recognize stop codons. 3. pecific sequences often occur near stop codon that aid in termination. F. General protein synthesis comments. 1. ma read 5' 3' 2. rotein synthesized from - to C-terminal end. 3. More than one ribosome can read an ma at a time. (Amplification!) 4. Very few errors occur. VIII. MUTATI, DA EAI & GEETIC DIEAE A. Mutations 1. A mutation is a change (fixed) in DA. 2. Mutation types a) silent (ex.: DA coded ma of UUU UUG) b) conservative [ma AUU (Ile) GUU (val)] c) ther. These cause substantial change in protein function. (ickle-cell anemia. Can you figure out the DA change from Glu Val?) 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? C 2 oxidative deamination U 5. Generally, mutagens are carcinogens and teratogens. UGA UGA C. Genetic Diseases occur when a mutation has a deleterious effect. 1. ickle-cell anemia ( a base substitution) 2. Xeroderma pigmentosum (thymine dimer repair) 13

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