Resources. This lecture Campbell and Farrell's Biochemistry, Chapter 12

Transcription

1 TRANSLATION 1

2 Resources This lecture Campbell and Farrell's Biochemistry, Chapter 12 2

3 The big question How is the nucleotide sequence of mrna translated into the amino acid sequence of a protein? 3

4 General information Protein synthesis involves interactions between three types of RNA molecules: trnas rrnas mrna templates 4

5 trna structure trnas are short RNA molecules (80 bases long) An amino acid is covalently attached to the ribose of the terminal adenosine charged or activated trna carries one amino acid Aminoacyl-tRNA synthetases 5

6 Codon vs. anticodon trnas also contain a three-nucleotide sequence known as anticodon that pairs with the codon or triplet mrna molecules 6

7 THE GENETIC CODE 7

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9 Features of the genetic Not universal Example: AUA In mitochondria, methionine In cytosol, isoleucine Wobble base pairing (degenrate codon) 9

10 Wobble base pairing 10

11 Prokaryotic vs. eukaryotic ribosomes 11

12 Enzymatic catalysis The large ribosomal subunit catalyzes the peptidyl transferase reaction The formation of a peptide bond is an RNA-catalyzed reaction 12

13 THE GENERAL MECHANISM OF TRANSLATION 13

14 Directionality Three stages: initiation, elongation, and termination The direction is 5 3 Protein synthesis begins at the amino terminus and extends toward the carboxyl terminus 14

15 Coupling of transcription and translation in prokaryotes Translation and transcription are coupled in space and time 15

16 Uncoupling of transcription and translation in eukaryotes Why? The nucleus mrna processing 16

17 Start of translation In both prokaryotes and eukaryotes, translation starts at specific initiation sites The 5 terminal portions upstream of the initiation sites of both prokaryotic and eukaryotic mrnas contain noncoding sequences, referred to as 5 untranslated regions 17

18 Remember Bacterial mrna is polycistronic Eukaryotic mrna is monocistronic 18

19 Shine-Delgarno sequence 19

20 But in eukaryotes Eukaryotic ribosomes recognize mrnas by binding to the 7-methylguanosine cap at their 5 terminus The ribosomes then scan downstream for the AUG initiation codon (Kozak sequence) 20

21 The first amino acid Translation always initiates with the amino acid methionine, usually encoded by AUG In most bacteria, it is N-formylmethionine 21

22 Translation initiation Three initiation factors (IF-1, IF-2, and IF-3) bind to the 30S ribosomal subunit IF-2: binding fmet-trna IF-3: binding of 50S ribosome 22

23 Translation elongation I 23

24 Translation termination 24

25 Selenocysteine UGA may code for selenocysteine, the twenty first amino acid The serine is converted enzymatically to selenocysteine Stem-loop structure 25

26 Polyribosomes (polysomes) 26

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28 Smooth vs. rough endoplasmic reticulum 28

29 Inhibitors of translation INHIBITOR Tetracycline SPECIFIC EFFECT blocks binding of aminoacyl-trna to A-site of ribosome Streptomycin Chloramphenicol Erythromycin prevents the transition from initiation complex to chainelongating ribosome blocks the peptidyl transferase reaction on ribosomes blocks the translocation reaction on ribosomes 29

30 Levels of regulation Transcription RNA processing RNA transport mrna stability Translation Post-translational modification Protein activity Protein degradation 30

31 Fate of (mis)- and (un)-folded proteins 31

32 Heme and protein synthesis In reticulocytes (immature erythrocytes), heme stimulates protein synthesis The mrna is translated only if adequate heme is available to form functional hemoglobin molecules 32

33 Regulation 33

34 ApoB-100 vs. apob-48 34

35 Synthesis of apob gene 35

36 Processing and mechanisms of action of microrna 36

37 mirnas and cancer Abnormalities in presence of mirnas have been linked with some types of cancer 37

38 Genomics and proteomics

39 Types of biomarkers Predictive biomarkers Diagnostics biomarker Therapeutic biomarkers Disease-activity biomarkers 39

40 Linkage disequilibrium When particular alleles at two different loci are inherited together more often than you would expect by chance, they are in Linkage disequilibrium (they map close together on the same chromosome) 40

41 Haplotype The set of alleles inherited together because they are linked is known as haplotype 41

42 HapMap A new strategy to identify the most useful, minimum set of SNP alleles among different populations that tag or label a haplotype block (so-called tag SNPs) Reduce scanning from 10 million SNPs to 500,000 42

43 Genome-wide association studies (GWAS) GWAS is an approach to identify genetic variants in a population hat are associated with a disease Requires large sample size, control samples be consistent with patient samples, confirmation by replication, and lots of statistics 43

44 Guilt by association 44

45 GWAS approaches Next-generation sequencing SNP microarray 45

46 Manhattan plot 46

47 How do different cells appear? All cells in one body have the same genome, but cell types in a multicellular organism become different from one another because they synthesize different sets of RNA and protein molecules The patterns of mrna can differentiate cell types from each other 47

48 Differential gene expression The cell types in a multicellular organism become different from one another because they synthesize and accumulate different sets of RNA and protein molecules At any one time, a typical human cell expresses approximately 10,000-20,000 of its approximately 30,000 genes 48

49 Important term The transcriptome (the total collection of RNA transcripts in a cell) 49

50 Northern blotting This is done exactly like Southern blotting except that RNA from cells is isolated instead of DNA Then RNA molecules are fractionated based on sizes by gel electrophoresis The fractionated RNA molecules are transferred to a membrane The RNA molecules on the membrane are targeted by a labeled DNA probe whose sequence is complementary to a specific RNA molecule 50

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53 In situ hybridization In situ hybridization methods reveals the distribution of specific RNA molecules in cells in tissues RNA molecules can hybridize when the tissue is incubated with a complementary DNA or RNA probe In this way the patterns of differential gene expression can be observed in tissues, and the location of specific RNAs can be determined in cells. 53

54 ISH procedure 54

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58 Limitations These techniques allow us to study the expression of a few genes It is important, however, to understand how the genome as a whole operates within the cell The determination of molecular networks is achieved by large-scale analysis of gene expression in cells 58

59 What is a DNA library? A library can be created for DNA fragments just like book libraries You can have clones of bacteria each containing a specific piece of DNA You can save these clones in the freezer and take whichever clone you want to study t/dnalibrary.html 59

60 Genomic DNA library 60

61 cdna library This library contains only those DNA sequences that are transcribed into mrna This is done by extracting the mrna from cells and then making a complementary DNA (cdna) copy of each mrna molecule present 61

62 cdna library 62

63 Differences between genomic and cdna libraries Genomic clones represent a random sample of all of the DNA sequences in an organism. By contrast, cdna clones contain only those regions of the genome that have been transcribed into mrna Because the cells of different tissues produce distinct sets of mrna molecules, a distinct cdna library is obtained for each type of cell used to prepare the library 63

64 The science of -omics 64

65 Studying the transcriptome as a whole One such method in studying transcriptomes is DNA microarrays, which allow the analysis of the RNA products of thousands of genes all at once By examining the expression of so many genes simultaneously, we can understand gene expression patterns in physiological and pathological states 65

66 DNA microarrays DNA microarrays are glass microscope slides spotted with up to tens of thousands of DNA fragments in an area the size of a fingernail The exact sequence and position of every DNA fragment on the array is known t/dnachips.html 66

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68 Using a DNA microarray mrna from the cells being studied is first extracted and converted to cdna The cdna is labeled with a radioactive probe The microarray is incubated with the labeled cdna sample for hybridization to occur If a gene is expressed, then the cdna will exist and bind to a specific complementary DNA fragment on the microarray Binding can be detected since the cdna is labeled and expression is determined 68

69 Comparative expression Typically the fluorescent cdnas from one sample (e.g. from cancer cells) are mixed with cdna fragments from another sample (e.g. normal cells) Both are labeled with two different fluorescent tags If the amount of RNA expressed from a particular gene in one sample is increased relative to that of the other sample, the color of it fluorescent tag will increase as well. We can then say that gene expression in that sample has increased relative to the other 69

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72 Expression biomarker The expression of a certain gene can be used as a biomarker of health status 72

73 Sample Sample Sample Combine results Eliminate samples 2, 4,& 9 Cluster samples according to expression Samples Genes

74 DNA microarrays and breast cancer 74

75 Basics of mass spectrometry Ion source Mass analyzer CID Mass analyzer HPLC Detector Detector 75

76 Types of protein microarrays Expression microarrays Y Y Y Y Y Y Y Y Direct labeling Forward-phase microarray Indirect labeling Forward-phase microarray Reverse-phase microarray Functional microarrays Interaction microarray Enzymatic microarray 76

77 Expression Array Probes (antibody) on surface recognize target proteins from 1-2 samples (antibody chip or forwardphase array) OR protein lysates from multiple samples arrayed on surface recognized by one-two antibodies (antigen chip or reverse-phase array) Identification of expressed proteins from samples Typical quantification method for large # of expressed proteins 77

78 Interaction Array Probes (proteins, peptides, lipids) on surface interact with target proteins Identification of protein interactions High throughput discovery of interactions 78

79 Functional Array Probes (proteins) on surface react with target molecules. Reaction products are detected 79

80 Interactome 80

81 THIS IS THE FUTURE OF MEDICINE 81

82 MOLECULAR BIOLOGY OF CANCER 82

83 Multi-step process 83

84 Cancer cells are clonal 84

85 Causes of cancer Hereditary Hormones (estrogen) Carcinogens Chemicals (aflatoxin) Radiation (X-ray) Pathogens Bacteria (H. pylori) Viruses (Human papiloma virus and hepatitis C) Parasites (bilharsza) 85

86 Abnormal balance Oncogenes Tumor suppressor genes 86

87 Balance of cell death/survival 87

88 Oncogenes A proto-oncogene is a gene whose protein product has the capacity to induce cellular transformation given it sustains some genetic insult. An oncogene is a gene that has sustained some genetic damage and, therefore, produces a protein capable of cellular transformation. Functions Cell proliferation Cell differentiation and Cell survival 88

89 Mutations of oncogenes Dominant Gain of function Activating point mutation Gene amplification or overexpression 89

90 Cell proliferation (Ras) 90

91 Cell differentiation (PML/RARα) 91

92 Cell survival (PI-3 kinase and Akt) 92

93 Tumor suppressor genes Regulation of progression of cell cycle Examples: Retinoblastoma (Rb) P53 93

94 Rb Nucleus cytoplasm 94

95 p53 95

96 Genetic model of colorectal cancer 96

97 Molecular diagnosis of cancer Estrogen receptor HER2 Brca1 BCR/ABL Ras FAP MLH1 and MSH2 97

98 Classification of tumor type by microarray analysis Prostate Bladder Breast Colorectal Gastroesophagus Kidney Liver Ovarian Pancreas Lung-adenocarcinoma Lung-squamous carcinoma 98

99 Triplenegative breast cancer 99

100 Hallmarks of cancer Increased release of growth factors Overexpression of receptors Mutational activation of signaling molecules Mutated tumor suppressor genes Reliance on glycolysis and lactate metabolism Release of immunosuppressive factors Recruitment of immunosuppressive cells Mutated tumor suppressor genes Mutator phenotype Low detection of genetic defects and mutations Telomerase Signals that induce proliferation, survival, angiogenesis, and invasion and metastasis Vascular endothelial growth factor Epithelial-mesenchymal transition (EMT) Decreased cell-cell adhesion Increased cell-matrix adhesion, proteolysis of ECM, and migration 100