TRANSLATION...•A proto-oncogene is a gene whose protein product has the capacity to induce...

TRANSLATION

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Resources

• This lecture

• Campbell and Farrell's Biochemistry, Chapter 12

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The big question

• How is the nucleotide sequence of mRNA translated into the amino acid sequence of a protein?

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General information

• Protein synthesis involves interactions between three types of RNA molecules:

– tRNAs

– rRNAs

– mRNA templates

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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

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Codon vs. anticodon

• tRNAs also contain a three-nucleotide sequence known as “anticodon” that pairs with the “codon’ or “triplet” mRNA molecules

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THE GENETIC CODE

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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

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Prokaryotic vs. eukaryotic ribosomes

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Enzymatic catalysis

• The large ribosomal subunit catalyzes the peptidyltransferase reaction

– The formation of a peptide bond is an RNA-catalyzed reaction

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THE GENERAL MECHANISM OF

TRANSLATION

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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

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Coupling of transcription and

translation in prokaryotes

• Translation and transcription are coupled in space and time

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Uncoupling of transcription and

translation in eukaryotes

• Why?

– The nucleus

– mRNA processing

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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

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Remember…

• Bacterial mRNA is polycistronic

• Eukaryotic mRNA is monocistronic

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Shine-Delgarno sequence

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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)

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The first amino acid

• Translation always initiates with the amino acid methionine, usually encoded by AUG

• In most bacteria, it is N-formylmethionine

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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

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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

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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

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Levels of regulation

• Transcription

• RNA processing

• RNA transport

• mRNA stability

• Translation

• Post-translational modification

• Protein activity

• Protein degradation

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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

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Synthesis of apoB gene

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Processing and mechanisms of

action of microRNA

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miRNAs and cancer

• Abnormalities in presence of miRNAs have been linked with some types of cancer

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Genomics and proteomics

Types of biomarkers

• Predictive biomarkers

• Diagnostics biomarker

• Therapeutic biomarkers

• Disease-activity biomarkers

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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)

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Haplotype

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

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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

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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

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“Guilt by association”

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GWAS approaches

• Next-generation sequencing

• SNP microarray

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Manhattan plot

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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

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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

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Important term

• The transcriptome (the total collection of RNA transcripts in a cell)

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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.

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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

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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

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Genomic DNA library

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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

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cDNA library

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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

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The science of -omics

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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

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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

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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

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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

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Expression biomarker

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

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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

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Combine resultsEliminate samples 2, 4,& 9Cluster samples according to expression

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DNA microarrays and breast cancer

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Ion

source

Mass

analyzer CID

Detector

Mass

analyzer

Detector

Basics of mass spectrometry

HPLC

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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

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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

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Interaction Array

• Probes (proteins, peptides, lipids) on surface interact with target proteins

• Identification of protein interactions

• High throughput discovery of interactions

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Functional Array

• Probes (proteins) on surface react with target molecules.

• Reaction products are detected

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Interactome

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THIS IS THE FUTURE OF MEDICINE

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MOLECULAR BIOLOGY OF CANCER

http://www.ncbi.nlm.nih.gov/books/NBK9840/

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Multi-step process

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Cancer cells are clonal

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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)

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Abnormal balance

Oncogenes

Tumor suppressor

genes

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Balance of cell death/survival

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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

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Mutations of oncogenes

• Dominant

• Gain of function

– Activating point mutation

– Gene amplification or overexpression

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Cell proliferation (Ras)

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Cell differentiation (PML/RARα)

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Cell survival (PI-3 kinase and Akt)

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Tumor suppressor genes

• Regulation of progression of cell cycle

• Examples:

– Retinoblastoma (Rb)

– P53

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Rb

Nucleus

cytoplasm94

p53

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Genetic model of colorectal cancer

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Molecular diagnosis of cancer

• Estrogen receptor

• HER2

• Brca1

• BCR/ABL

• Ras

• FAP

• MLH1 and MSH2

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Prostate

Bladder

Breast

Colorectal

Gastroesophagus

Kidney

Liver

Ovarian

Pancreas

Lung-adenocarcinoma

Lung-squamous carcinoma

Classification of tumor type by

microarray analysis

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Triple-negative breast cancer

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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

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