DNA mrna Protein (genome) (transcriptome) (proteome)

Transcription

1 hapter 28: ucleosides, ucleotides, and ucleic cids. ucleic acids are the third class of biopolymers (polysaccharides and proteins being the others) wo major classes of nucleic acids deoxyribonucleic acid (D): carrier of genetic information ribonucleic acid (R): an intermediate in the expression of genetic information and other diverse roles he entral Dogma (F. rick): D mr Protein (genome) (transcriptome) (proteome) he monomeric units for nucleic acids are nucleotides ucleotides are made up of three structural subunits 1. Sugar: ribose in R, 2-deoxyribose in D 2. eterocyclic base 3. Phosphate 340 ucleoside, nucleotides and nucleic acids phosphate sugar base phosphate phosphate sugar base sugar base sugar base nucleoside nucleotides phosphate sugar base nucleic acids he chemical linkage between monomer units in nucleic acids is a phosphodiester

2 28.1: Pyrimidines and Purines. he heterocyclic base; there are five common bases for nucleic acids (able 28.1, p. 1166). ote that, and exist in the keto form (and not the enol form found in phenols) purine pyrimidine 2 cytosine () D/R 2 adenine () D/R 3 thymine () D 2 guanine () D/R uracil () R 28.2: ucleosides. -lycosides of a purine or pyrimidine heterocyclic base and a carbohydrate. he - bond involves the anomeric carbon of the carbohydrate. he carbohydrates for nucleic acids are D-ribose and 2-deoxy-D-ribose 342 ucleosides = carbohydrate + base (able 28.2, p. 1168) ribonucleosides or 2 -deoxyribonucleosides ' 1' 2' X R: X=, adenosine () D: X=, 2'-deoxyadenosine (d) ' 1' 2' X R: X=, cytidine () D: X=, 2'-deoxycytidine (d) 2 X 2 R: X=, guanosine () D: X=, 2'-deoxyguanosine (d) 3 D: thymidine () R: R=, uridine () o differentiate the atoms of the carbohydrate from the base, the position number of the carbohydrate is followed by a (prime). he stereochemistry of the glycosidic bond found in nucleic acids is β

3 28.3: ucleotides. Phosphoric acid esters of nucleosides. ucleotides = nucleoside + phosphate B P B X X ribonucleoside (X=) deoxyribonucleoside (X=) ribonucleotide -monophosphate (X=, MP) deoxyribonucleotide -monophosphate (X=, dmp) P P ribonucleotide -diphosphate (X=, DP) deoxyribonucleotide -diphosphate (X=, ddp) X B P P P ribonucleotide -triphosphate (P) deoxyribonucleotide -triphosphate (X=, dp) X B P B ribonucleotide,-cyclic phosphosphate (cmp) Kinase: enzymes that catalyze the phosphoryl transfer reaction from P to an acceptor substrate. M 2+ dependent lucose P P P P 2 P 3 2- lucose-6-phosphate P P DP P P P P P P P P 2 adenylyl cyclase -P 2 7 P cmp 2 2 P guanylate cyclase 2 P -P 2 7 P cmp MP MP : Bioenergetics. (Please read) 28.5: P and Bioenergetics. (Please read) 1971 obel Prize in Medicine or Physiology: Earl Sutherland P P P P 2 2 P P DP 2 + P ~31 KJ/mol (7.4 Kcal/mol)

4 28.6: Phosphodiesters, ligonucleotides, and Polynucleotides. he chemical linkage between nucleotide units in nucleic acids is a phosphodiester, which connects the 5 -hydroxyl group of one nucleotide to the 3 -hydroxyl group of the next nucleotide. By convention, nucleic acid sequences are written from left to right, from the 5 -end to the 3 -end. P P ucleic acids are negatively charged R, X= D, X= 28.7: ucleic cids. 1944: very, MacLeod & Mcarty - Strong evidence that D is genetic material 1950: hargaff - careful analysis of D from a wide variety of organisms. ontent of,, & varied widely according to the organism, however: = and = (hargaff Rule) 1953: Watson & rick - structure of D (1962 obel Prize with 346 M. Wilkens, 1962) Base X Base X 28.8: Secondary Structure of D: he Double elix. Initial like-with-like, parallel helix: Does not fit with with hargaff s Rule: = = purine - purine pyrimidine - pyrimidine Wrong tautomers!! Watson, J. D. he Double elix,

5 wo polynucleotide strands, running in opposite directions (anti-parallel) and coiled around each other in a double helix. he strands are held together by complementary hydrogenbonding between specific pairs of bases. ntiparallel - Pair ydrogen Bond Donor 2 ydrogen Bond 3 cceptor ydrogen Bond cceptor Bond 6 ydrogen cceptor 4 R 1 ydrogen Bond R Donor ydrogen Bond 2 Donor R R ntiparallel - Pair ydrogen Bond cceptor 4 ydrogen Bond 3 Donor R 6 ydrogen Bond Donor R 1 ydrogen Bond cceptor R R 348 D double helix major groove 12 Å one helical turn 34 Å minor groove 6Å backbone: deoxyribose and phosphodiester linkage bases

6 28.9: ertiary Structure of D: Supercoils. Each cell contains about two meters of D. D is packaged by coiling around a core of proteins known as histones. he D-histone assembly is called a nucleosome. istones are rich is lysine and arginine residues. Pdb code 1kx5 350 It has not escaped our attention that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. Watson & rick 28.10: Replication of D. he entral Dogma (F. rick): D replication D transcription mr translation Protein (genome) (transcriptome) (proteome) Expression and transfer of genetic information: Replication: process by which D is copied with very high fidelity. ranscription: process by which the D genetic code is read and transferred to messenger R (mr). his is an intermediate step in protein expression ranslation: he process by which the genetic code is converted to a protein, the end product of gene expression. he D sequence codes for the mr sequence, which codes for the protein sequence

7 D is replicated by the coordinated efforts of a number of proteins and enzymes. For replication, D must be unknotted, uncoiled and the double helix unwound. opoisomerase: Enzyme that unknots and uncoils D elicase: Protein that unwinds the D double helix. D polymerase: Enzyme that replicates D using each strand as a template for the newly synthesized strand. D ligase: enzyme that catalyzes the formation of the phosphodiester bond between pieces of D. D replication is semi-conservative: Each new strand of D contains one parental (old, template) strand and one daughter (newly synthesized) strand 352 nwinding of D by helicases expose the D bases (replication fork) so that replication can take place. elicase hydrolyzes P in order to break the hydrogen bonds between D strands D replication

8 D Polymerase: the new strand is replicated from the 5 3 (start from the 3 -end of the template) D polymerases are Mg 2+ ion dependent he deoxynucleotide 5 -triphosphate (dp) is the reagent for nucleotide incorporation template strand (old) P - P - P P dp Mg 2+ (new) 3 -hydroxyl group of the growing D strand acts as a nucleophile and attacks the α-phosphorus atom of the dp. 354 Replication of the leading strand occurs continuously in the 5 3 direction of the new strand. Replication of the lagging strand occurs discontinuously. Short D fragments are initially synthesized and then ligated together. D ligase catalyzes the formation of the phosphodiester bond between pieces of D. animations of D processing:

9 D replication occurs with very high fidelity: Most D polymerases have high intrinsic fidelity Many D polymerases have proof-reading (exonuclease) activity Mismatch repair proteins seek out and repair base-pair mismatches due to unfaithful replication Ribonucleic cid R contains ribose rather than 2-deoxyribose and uracil rather than thymine. R usually exist as a single strand. here are three major kinds of R messenger R (mr): ribosomal R (rr) transfer R (tr) D is found in the cell nucleus and mitochondria; R is more 356 disperse in the cell. ranscription: only one of the D strands is copied (coding or antisense strand). n R polymerase replicates the D sequence into a complementary sequence of mr (template or sense strand). mrs are transported from the nucleus to the cytoplasm, where they acts as the template for protein biosynthesis (translation). three base segment of mr (codon) codes for an amino acid.he reading frame of the codons is defined by the start and stop codons

10 -cap -R start stop -R oding sequence he mr is positioned in the ribosome through complementary pairing of the 5 -untranslated region of mr with a rr. ransfer R (tr): t-rs carries an amino acid on the 3 -terminal hydroxyl () (aminoacyl t-r) and the ribosome catalyzes amide bond formation. Ribosome: large assembly of proteins and rrs that catalyzes protein and peptide biosynthesis using specific, complementary, anti-parallel pairing interactions between mr and the anti-codon loop of specific tr s. 358 lthough single-stranded, there are complementary sequences within tr that give it a defined conformation. he three base codon sequence of mr are complementary to the anti-codon loops of the appropriate tr. he basepairing between the mr and the tr positions the trs for amino acid transfer to the growing peptide chain. variable loop ψ loop D loop aminoacyl t-r

11 28.12: Protein Biosynthesis. Ribosomal protein synthesis S 3 S 3 2 P site site S 3 3S 3S J 2 3 E site : IDS. (please read) 28.14: D Sequencing. Maxam-ilbert: relys on reagents that react with a specific D base that can subsequent give rise to a sequence specific cleavage of D Sanger: Enzymatic replication of the D fragment to be sequenced with a D polymerase, Mg +2, and dideoxynucleotides triphosphate (ddp) that truncates D replication Restriction endonucleases: Bacterial enzymes that cleave D at specific sequences 5 -d(-----)-3 3 -d(-----)-5 EcoR I restriction enzyme 5 -d(-----)-3 3 -d(-----)-5 BM I 2-3P 2-3P

12 Sanger Sequencing: key reagent: dideoxynucleotides triphosphates (ddp) P P P P P P P P P P P P ddp ddp ddp ddp - 32 P primer D polymerase Mg 2+, dp's + one ddp When a ddp is incorporated elongation of the primer is terminated he ddp is specifically incorporated opposite its complementary nucleotide base nti-viral ucleosides 3 Z ddi 2 2 S d4 (-)-3 emplate unknown sequence 362 Sanger Sequencing Larger fragments Smaller fragments dd dd dd dd 32 P 32 P- primer template

13 28.15: he uman enome Project. (please read) 28.16: D Profiling and Polymerase hain Reaction (PR). method for amplifying D using a D polymerase, dps and cycling the temperature. eat stable D Polymerases (from archaea): aq: thermophilic bacteria (hot springs)- no proof reading Pfu: geothermic vent bacteria- proof reading Mg 2+ two Primer D strands (synthetic, large excess) one sense primer and one antisense primer one emplate D strand (double strand) dp s 1 x 2 = 2 x 2 = 4 x 2 = 8 x 2 = 16 x 2 = 32 x 2 = 64 x 2 = 128 x 2 = 256 x 2 = 512 x 2 = 1,024 x 2 = 2,048 x 2 = 4,096 x 2 = 8,192 x 2 = 16,384 x 2 = 32,768 x 2 = 65,536 x 2 = 131,072 x 2 = 262,144 x 2 = 524,288 x 2 = 1,048,576 In principle, over one million copies per original, can be obtained after just twenty cycles KRY B. MLLIS, 1993 obel Prize in hemistry for his invention of the polymerase chain reaction (PR) method. 364 Polymerase hain Reaction 95 denaturation anneal (+) and (-) primers 72 aq, Mg 2+, dps extension 95 denaturation 2nd cycle 2 copies of D repeat temperature cycles amplification of D For a PR animation go to: