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Basic Molecular Biology

Basic Molecular Biology. January 2002. Macromolecules and their unit components. Macro molecules unit components Nucleic acids nucleotides (DNA& RNA) Proteins amino acids Carbohydrates Sugars Lipids Fatty acids. √. √. √. O. H. +. H. -. N. C. H. C. O. H. pH 6-7. R.

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Basic Molecular Biology

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  1. Basic Molecular Biology January 2002

  2. Macromolecules and their unit components Macro molecules unit components Nucleic acids nucleotides (DNA& RNA) Proteinsamino acids CarbohydratesSugars LipidsFatty acids √ √ √

  3. O H + H - N C H C O H pH 6-7 R Amino acids O H H N C amine group carboxyl H C OH R side chain

  4. CH 3 CH CH 2 CH 3 CH 3 O C CH 2 - O Amino acids groups Group Characteristics Names Example (-Rx) non-polar hydrophobic Ala, Val, Leu, Ile, Pro, Phe Trp, Met polar hydrophilic Gly , Ser, Thr, (non-charged) Cys, Tyr, Asn Gln acidic negatively Asp, Glu charged basic positively Lys, Arg, His charged Leu OH CH Thr Asp Total = 20 Lys

  5. CH 2 SH CH 2 CH 2 C O- O peptides have different sequences OH CH O O - CH CH C O + NH NH C 3 NH C NH C CH CH O O CH 2 OH Tyr------Cys--------Asp-----------Ser = Y-C-D-S

  6. Primary structure The primary structure of a protein is defined as its sequence of amino acids. Connexin26 MDWGTLQSIL GGVNKHSTSI GKIWLTVLFI FRIMILVVAA KEVWGDEQAD FVCNTLQPGC KNVCYDHHFP ISHIRLWALQ LIMVSTPALL VAMHVAYRRH EKKRKFMKGE IKNEFKDIEE IKTQKVRIEG SLWWTYTTSI FFRVIFEAVF MYVFYIMYNG FFMQRLVKCN AWPCPNTVDC FISRPTEKTV FTVFMISVSG ICILLNITEL CYLFVRYCSG KSKRPV

  7. Questions: How is the information about protein sequences - stored in the cell ? - duplicated after cell division? Answer: ...DNA...

  8. NH 2 N N BASE N N OH OH OH P O CH OH P O P O 2 O RIBOSE SUGAR O O O TRIPHOSPHATE OH NUCLEOTIDE

  9. ATP GTP TTP (UTP) CTP O NH NH 2 2 N N CH N 3 H N 2 N N O N OH OH OH P O CH OH P O P O 2 O OH OH OH O O O P O CH OH P O P O 2 O O O O OH OH OH OH Nucleotides PurinesPyrimidines

  10. 5’ CH 2 4’ 1’ 3’ 2’ 5’ CH 2 4’ 1’ 3’ 2’ 5’ CH 2 4’ 1’ 3’ 2’ base Nucleotide chain 5’ end OH OH OH P O OH P O P O O N O O O O base O OH OH OH O HO P OH P O P O O O O O base O OH O HO P O O 3’ end OH OH

  11. CH 3 Nucleotide Pairing H- bonds O H H N N H N N N O N Thymidine N Adenine T A

  12. CH 3 H CH 3 N H N N O Nucleotide Pairing H- bonds O H H N N H N N N H- bonds O N Thymidine N O Adenine T A H N N Cytosine N H N N H C G Guanine

  13. A G T C The double helix G A A 5’ACGGGTACATGAC3’ ||||||||||||| 3’TGCCCATGTACTG5’ C T T G C T A T A antiparallel complimentary strand A T C G G C A G T C

  14. Types of Nucleic Acids DNA nuclear DNA (linear) mitochondrial DNA (circular) plasmid DNA (circular RNA tRNA (transfer RNA) rRNA (ribosomal RNA) hnRNA (heteronuclear RNA) mRNA (messenger)

  15. CH CH 2 2 Differences between RNA and DNA RNA DNA O O 1) ribose sugar OH OH OH (ribonucleic acid) (deoxy ribonucleic acid)

  16. CH CH 2 2 CH 3 Differences between RNA and DNA RNA DNA O O 1) ribose sugar OH OH OH (ribonucleic acid) (deoxy ribonucleic acid) O O 2 )T and U N N O O N N uracyl thymidine

  17. CH CH 2 2 CH 3 Differences between RNA and DNA RNA DNA O O 1) ribose sugar OH OH OH (ribonucleic acid) (deoxy ribonucleic acid) O O 2 )T and U N N O O N N uracyl thymidine 3) strand single double

  18. DNA replication GGG 3’ ATGGGTACA CATGAC 5’TGAC |||| 3’ACTG ||| ||||||||||||||| GTACTG CCC 5’ TACCCATGT

  19. CATGAC ATGGGTACA GGG 3’ 5’TGAC |||| 3’ACTG G ||| A CCC 5’ TACCCATGT GTACTG DNA replication DNA polymerase

  20. CATGAC ATGGGTACA T GGG 3’ 5’TGAC |||| 3’ACTG G ||| A T CCC 5’ TACCCATGT GTACTG DNA replication

  21. CATGAC ATGGGTACA C T GGG 3’ 5’TGAC |||| 3’ACTG G ||| A T CCC 5’ G TACCCATGT GTACTG DNA replication

  22. CATGAC ATGGGTACA A C T GGG 3’ 5’TGAC |||| 3’ACTG G ||| A T CCC 5’ G G TACCCATGT GTACTG DNA replication

  23. DNA replication ACGGGTACA CATGAC GGG 3’ 5’ACGGGTACATGAC ||||||||||||| 3’TGCCCATGTACTG 3’ACTG5’ ||| CCC 5’ 5’ACGGGT3’ GTACTG TGCCCATGT 5’ACGGGTACATGAC ||||||||||||| 3’TGCCCATGTACTG ACGGGTAC 3’ |||||||| TGCCCATG 5’ old new semiconservative replication 5’ACGGGTACATGAC ||||||||||||| 3’TGCCCATGTACTG ACGGGTAC 3’ |||||||| TGCCCATG 5’ new old

  24. DNA proof-reading CATGAC ATGGGTACA A C T GGG 3’ 5’TGAC |||| 3’ACTG G ||| A T G CCC 5’ T TACCCATGT GTACTG DNA polymerase has exonuclease activity: it can cut out unpaired bases to prevent mistakes(mutations) in DNA replication

  25. DNA proof-reading CATGAC ATGGGTACA A C T GGG 3’ 5’TGAC |||| 3’ACTG G ||| A T G CCC 5’ T TACCCATGT GTACTG DNA polymerase has exonuclease activity: it can cut out unpaired bases to prevent mistakes(mutations) in DNA replication

  26. DNA proof-reading CATGAC ATGGGTACA A C T GGG 3’ 5’TGAC |||| 3’ACTG G T ||| A T G CCC 5’ G TACCCATGT GTACTG DNA polymerase has exonuclease activity: it can cut out unpaired bases to prevent mistakes(mutations) in DNA replication

  27. DNA is located in the cell nucleus • DNA is associated with histones protein evenly distributed approximately every 200bp. (DNA+histones =chromatin) • DNA subdivided in chromosomes whose number differ among species (46 in humans, 22+22+X +Y) 38 44 46 Number of chromosomes DNA(basic facts)

  28. + N H 3 A R G 1 T C G A A C T T R 2 G C T A T A R 3 A T C G G C A R G 4 T C - O From DNA to protein C H O C N H CH O C N H C H O C N H C H O C

  29. DNA ACGTCTCAA TGCAGAGTT RNA polymerase nuclear factors Transcription RNA ACGUCUCAA ribosomes (proteins+ rRNA) t RNAs Translation Protein Thr-Ser-Gln Protein synthesis

  30. Transcription GGG 3’ ATGGGTACA CATGAC 5’TGAC |||| 3’ACTG ||| ||||||||||||||| GTACTG CCC 5’ TACCCATGT

  31. 5’ TGACATGGGTACACATGACGGG 5’ ACTGTACCCATGTGTACTGCCC 3’ RNA polymerase Transcription 3’ U G A U A U

  32. CYTOSOL NUCLEUS UGAUAUAAAA MESSANGER RNA (mRNA) Transcription UGAUAU AAAA

  33. anticodon UAC transfer RNA (tRNA) there are 20 different tRNA (each one carrying a specific amino acid) Met Translation(from mRNA to protein) Triplet codon: three nucleotides code for one amino acid AUG = Met ACA = Thr CAU = His UUU = Phe

  34. Translation AUG ACG UCU CAA mRNA

  35. UAC Met Translation AUG ACG UCU CAA mRNA

  36. UAC UGC Met Thr Translation AUG ACG UCU CAA mRNA

  37. UGC AGA Thr Ser Translation AUG ACG UCU CAA mRNA Met

  38. UGC Thr Translation AUG ACG UCU CAA mRNA GUU Met Ser Gln

  39. Translation Ribosome mRNA AUGAUAGCCGAUU

  40. Translation Ribosome AUGAUAGCCGAUU

  41. Translation Ribosome AUGAUAGCCGAUU N-terminus growing protein C-terminus

  42. Start and Stop codons ACCA-AUG-AUA-GCC-GAU-GGG-UGA-GGAG The start codon is AUG and it also codes for Methionine There are three stop codons UGA, UAA and UAG

  43. Codon Degeneracy -there are 20 amino acids but 64 codons -for some amino acid there is more than one codon -the last of the three bases is the least specific CGU CGC CGG Arg CGA AGA AGG UUU UUA UUG UUC CAA CAG Phe Gln AUG Met UGG Trp

  44. Post-translational modification After synthesis a protein can undergo one or more modifications. e.g. • Tertiary structure : regulated by chaperons • Glycosidation: addition of sugars • Proteolitic cleavage • Phosphorylation: addition of phosphate groups to Tyr, Thr and Ser. • Other....

  45. THE MUTATIONS • AGTFTHEDOGATETHECATANDTHEBUNENDAFAT AGTF THE DOG ATE THE CAT AND THE BUN END AFAT AGTF THE DOG ATE THH CAT AND THE BUN END AFAT AGTF THE HOG ATE THE CAT AND THE BUN END AFAT AGTF THE DOG ATE THE CAT END THEBUNENDAFAT AGTF THE DOP GAT ETH ECA TAN DTH EBU NEN DAF AT AGTF THE DOG ATE THC ATA NDT HEB UNE NDA FAT AGTF THE DOG ATE CAT AND THE BUN END AFAT E

  46. MUTATIONS • UGUAC AUG UAU ACG UCU CAA UGA UCCA Met Tyr Ser Thr Gln STOP POINT MUTATIONS • UGUAC AUG UAU ACG UCU CAG UGA UCCA Met Tyr Ser Thr Gln STOP • UGUAC AUG UAU ACG CCU CAA UGA UCCA Met Tyr Ser Pro Gln STOP • UGUAC AUG UAA ACG UCU CAA UGA UCCA Met STOP

  47. MUTATIONS A • UGUAC AUG UAU ACG UCU CAA UGA UCCA Met Tyr Ser Thr Gln STOP Deletions • UGUAC AUG UAU CGU CUC AAU GAU CCA Met Tyr Arg Leu Asn Asp Pro • UGUAC AUG UAU UCU CAA UGA UCCA Met Tyr Thr Gln STOP Insertion • UGUAC AUG UAU ACG AUC UCA AUG AUC Met Tyr Ser Ile Ser Met Ile ACG A

  48. AATTCGCATGATGCATGCTCGAGCATAGC GCGTACTACGTACGAGCTCGTATCG ACGTGCCATG TGCACGGTACTTAA Restriction enzymes Enzymes found in bacteria that have the ability to cut DNA at specific sites EcoRI (GATTC) ACGTGCCATGAATTCGCATGATGCATGCTCGAGCATAGC TGCACGGTACTTAAGCGTACTACGTACGAGCTCGTATCG

  49. EcoRI (GATTC) XhoI (CTCGAG) AATTCGCATGATGCATGCTCG GCGTACTACGTACGA ACGTGCCATG TGCACGGTACTTAA AGCATAGC GCTCGTATCG Restriction enzymes ACGTGCCATGAATTCGCATGATGCATGCTCGAGCATAGC TGCACGGTACTTAAGCGTACTACGTACGAGCTCGTATCG

  50. recombination ACGTGCCATGAATTCGCATCATGCGAATTCATAGC TGCACGGTACTTAAGCGTACTACGCTTAAGTATCG TAGCATGAATTCGCATCGATC ATCGTACTTAAGCGTAGCTAG PLASMID

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