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TRANSLATION
1
Resources
• This lecture
• Campbell and Farrell's Biochemistry, Chapter 12
2
The big question
• How is the nucleotide sequence of mRNA translated into the amino acid sequence of a protein?
3
General information
• Protein synthesis involves interactions between three types of RNA molecules:
– tRNAs
– rRNAs
– mRNA templates
4
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
Codon vs. anticodon
• tRNAs also contain a three-nucleotide sequence known as “anticodon” that pairs with the “codon’ or “triplet” mRNA molecules
6
THE GENETIC CODE
7
8
Features of the genetic
• Not universal
– Example: AUA
• In mitochondria, methionine
• In cytosol, isoleucine
• Wobble base pairing (degenrate codon)
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Wobble base pairing
10
Prokaryotic vs. eukaryotic ribosomes
11
Enzymatic catalysis
• The large ribosomal subunit catalyzes the peptidyltransferase reaction
– The formation of a peptide bond is an RNA-catalyzed reaction
12
THE GENERAL MECHANISM OF
TRANSLATION
13
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
Coupling of transcription and
translation in prokaryotes
• Translation and transcription are coupled in space and time
15
Uncoupling of transcription and
translation in eukaryotes
• Why?
– The nucleus
– mRNA processing
16
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
Remember…
• Bacterial mRNA is polycistronic
• Eukaryotic mRNA is monocistronic
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Shine-Delgarno sequence
19
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
The first amino acid
• Translation always initiates with the amino acid methionine, usually encoded by AUG
• In most bacteria, it is N-formylmethionine
21
Translation initiation
• Three initiation factors (IF-1, IF-2, and IF-3) bind to the 30S ribosomal subunit
• IF-2: binding fMet-tRNAIF-3: binding of 50S ribosome
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Translation elongation I
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Translation termination
24
Selenocysteine
• UGA may code for selenocysteine, the twenty first amino acid– The serine is converted enzymatically to selenocysteine
Stem-loop structure
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Polyribosomes (polysomes)
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Smooth vs. rough endoplasmic
reticulum
28
Inhibitors of translation
SPECIFIC EFFECTINHIBITOR
blocks binding of aminoacyl-tRNA to A-site of ribosomeTetracycline
prevents the transition from initiation complex to chain-
elongating ribosome
Streptomycin
blocks the peptidyl transferase reaction on ribosomesChloramphenicol
blocks the translocation reaction on ribosomesErythromycin
29
Levels of regulation
• Transcription
• RNA processing
• RNA transport
• mRNA stability
• Translation
• Post-translational modification
• Protein activity
• Protein degradation
30
Fate of (mis)- and (un)-folded proteins
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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
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Regulation
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ApoB-100 vs. apoB-48
34
Synthesis of apoB gene
35
Processing and mechanisms of
action of microRNA
36
miRNAs and cancer
• Abnormalities in presence of miRNAs have been linked with some types of cancer
37
Genomics and proteomics
Types of biomarkers
• Predictive biomarkers
• Diagnostics biomarker
• Therapeutic biomarkers
• Disease-activity biomarkers
39
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
Haplotype
• The set of alleles inherited together because they are linked is known as haplotype
41
HapMap
• A new strategy to identify the most useful, minimumset 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
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
“Guilt by association”
44
GWAS approaches
• Next-generation sequencing
• SNP microarray
45
Manhattan plot
46
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
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
Important term
• The transcriptome (the total collection of RNA transcripts in a cell)
49
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
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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
ISH procedure
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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
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
• http://www.sumanasinc.com/webcontent/animations/content/dnalibrary.html
59
Genomic DNA library
60
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
cDNA library
62
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
The science of -omics
64
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
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
• http://learn.genetics.utah.edu/content/labs/microarray/
• http://www.sumanasinc.com/webcontent/animations/content/dnachips.html
66
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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
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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Expression biomarker
• The expression of a certain gene can be used as a biomarker of health status
72
1 2 3
4 5 6
7 8 9
Sample 1
1 2 3
4 5 6
7 8 9
Sample 2
1 2 3
4 5 6
7 8 9
Sample 3
Samples1 3 2
Genes
1
5
3
8
7
6
Combine resultsEliminate samples 2, 4,& 9Cluster samples according to expression
73
DNA microarrays and breast cancer
74
Ion
source
Mass
analyzer CID
Detector
Mass
analyzer
Detector
Basics of mass spectrometry
HPLC
75
Types of protein microarrays
YY
Expression microarrays
Functional microarraysYY
YY
Y Y
Direct labeling Forward-phase microarray
Indirect labeling Forward-phase microarray
Reverse-phase microarray
Interaction microarray Enzymatic microarray
76
Expression Array
• Probes (antibody) on surface recognize target proteins from 1-2 samples (antibody chip or forward-phase 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
Interaction Array
• Probes (proteins, peptides, lipids) on surface interact with target proteins
• Identification of protein interactions
• High throughput discovery of interactions
78
Functional Array
• Probes (proteins) on surface react with target molecules.
• Reaction products are detected
79
Interactome
80
THIS IS THE FUTURE OF MEDICINE
81
MOLECULAR BIOLOGY OF CANCER
http://www.ncbi.nlm.nih.gov/books/NBK9840/
82
Multi-step process
83
Cancer cells are clonal
84
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
Abnormal balance
Oncogenes
Tumor suppressor
genes
86
Balance of cell death/survival
87
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
Mutations of oncogenes
• Dominant
• Gain of function
– Activating point mutation
– Gene amplification or overexpression
89
Cell proliferation (Ras)
90
Cell differentiation (PML/RARα)
91
Cell survival (PI-3 kinase and Akt)
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Tumor suppressor genes
• Regulation of progression of cell cycle
• Examples:
– Retinoblastoma (Rb)
– P53
93
Rb
Nucleus
cytoplasm94
p53
95
Genetic model of colorectal cancer
96
Molecular diagnosis of cancer
• Estrogen receptor
• HER2
• Brca1
• BCR/ABL
• Ras
• FAP
• MLH1 and MSH2
97
Prostate
Bladder
Breast
Colorectal
Gastroesophagus
Kidney
Liver
Ovarian
Pancreas
Lung-adenocarcinoma
Lung-squamous carcinoma
Classification of tumor type by
microarray analysis
98
Triple-negative breast cancer
99
Hallmarks of cancer
Increased release of growth factorsOverexpression of receptorsMutational activation of signaling molecules
Signals that induce proliferation, survival, angiogenesis, and invasion and metastasis
Mutated tumor suppressor genes
Mutated tumor suppressor genes
Telomerase
Vascular endothelial growth factor
Epithelial-mesenchymal transition (EMT)Decreased cell-cell adhesionIncreased cell-matrix adhesion, proteolysis of ECM, and migration
Mutator phenotypeLow detection of genetic defects and mutations
Release of immunosuppressive factorsRecruitment of immunosuppressive cells
Reliance on glycolysisand lactate metabolism
100