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Gene expression concept and analysis


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gene expression & comparison between PCR / RT-PCR

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Gene expression concept and analysis

  1. 1. GeneExpression:ConceptandAnalysis Noha Lotfy Ibrahim
  2. 2. The Central Dogma • Proposed by Francis Crick 1958 • DNA holds the coded hereditary information in the nucleus • This code is expressed at the ribosome during protein synthesis in the cytoplasm • The protein produced by the genetic information is what is influenced by natural selection • If a protein is modified it cannot influence the gene that codes for it • Therefore there is one way flow of information: DNA(transcription)  RNA(translation)  Protein
  3. 3. The central dogma biology
  4. 4. Gene Structure  Gene is the sequence of nucleotides in DNA encoding for one mRNA molecule or one polypeptide chain.  Eukaryotic gene structure: Most eukaryotic genes in contrast to typical bacterial genes, the coding sequences (exons) are interrupted by noncoding DNA (introns). The gene must have (Exon; start signals; stop signals; regulatory control elements).  The average gene 7-10 exons spread over 10-16kb of DNA.
  5. 5. Deoxy ribonuclic acid of DNA • DNA is a very stable molecule • It is a good medium for storing genetic material but… • DNA can do nothing for itself • It requires enzymes for replication • It requires enzymes for gene expression • The information in DNA is required to synthesise enzymes (proteins) but enzymes are require to make DNA function
  6. 6. RIBONUCLEIC ACID (RNA) •Found all over the cell (nucleus, mitochondria, chloroplasts, ribosomes and the soluble part of the cytoplasm). •Certain forms of RNA have catalytic properties •RIBOZYMES •Ribosomes and snRNPs are ribozymes •RNA could have been the first genetic information synthesizing proteins… •…and at the same time a biocatalyst •Reverse transcriptase provides the possibility of producing DNA copies from RNA
  7. 7. Types • Messenger RNA (mRNA) <5% • Ribosomal RNA (rRNA) Up to 80% • Transfer RNA (tRNA) About 15% • In eukaryotes small nuclear ribonucleoproteins (snRNP).
  8. 8. Structural characteristics of RNA molecules • Single polynucleotide strand which may be looped or coiled (not a double helix) • Sugar Ribose (not deoxyribose) • Bases used: Adenine, Guanine, Cytosine and Uracil (not Thymine).
  9. 9. mRNA • A long molecule 1 million Daltons • Ephemeral • Difficult to isolate • mRNA provides the plan for the polypeptide chain
  10. 10. rRNA • Coiled • Two subunits: a long molecule 1 million Daltons a short molecule 42 000 Daltons • Fairly stable • Found in ribosomes • Made as subunits in the nucleolus • rRNA provides the platform for protein synthesis
  11. 11. tRNA • Short molecule about 25 000 Daltons • Soluble • At least 61 different forms each has a specific anticodon as part of its structure. • tRNA “translates” the message on the mRNA into a polypeptide chain
  12. 12. Gene Expression Concept
  13. 13. Gene Expression  The process by which a gene's information is converted into the structures and functions of a cell by a process of producing a biologically functional molecule of either protein or RNA (gene product) is made (from genotype to phenotype). • Gene expression is assumed to be controlled at various points in the sequence leading to protein synthesis. • Idea: measuring amount of mRNA to see which genes are expressed, as protein measuring is more difficult.
  14. 14. Gene Expression Transcription  Synthesis of mRNA that is complementary to one of the strands of DNA. This happens in the nucleus of eukaryotes. Translation  Ribosomes synthesize a polypeptide chain using the genetic code on the mRNA molecule as its guide and make protein according to its instruction.
  15. 15. Transcription plan Transcription DNA messenger RNA Gene Nucleus
  16. 16. Transcription 17
  17. 17. Transcription: The synthesis of a strand of mRNA (and other RNAs) • Uses an enzyme RNA polymerase • Proceeds in the same direction as replication (5’ to 3’) • Forms a complementary strand of mRNA • It begins at a promotor site which signals the beginning of gene is not much further down the molecule (about 20 to 30 nucleotides) • After the end of the gene is reached there is a terminator sequence that tells RNA polymerase to stop transcribing NB Terminator sequence ≠ terminator codon.
  18. 18. Editing the mRNA • In prokaryotes the transcribed mRNA goes straight to the ribosomes in the cytoplasm • In eukaryotes the freshly transcribed mRNA in the nucleus is about 5000 nucleotides long • When the same mRNA is used for translation at the ribosome it is only 1000 nucleotides long • The mRNA has been edited • The parts which are kept for gene expression are called EXONS (exons = expressed) • The parts which are edited out (by snRNP molecules) are called INTRONS. © 2010 Paul Billiet ODWS
  19. 19. Transcription Enzymes RNA polymerase: The enzyme that controls transcription and is characterized by:  Search DNA for initiation site,  It unwinds a short stretch of double helical DNA to produce a single-stranded DNA template,  It selects the correct ribonucleotide and catalyzes the formation of a phosphodiester bond,  It detects termination signals where transcript ends. 20
  20. 20. Transcription Enzymes Polymerase I nucleolus Makes a large precursor to the major rRNA (5.8S,18S and 28S rRNA in vertebrates Polymerase II nucleoplasm Synthesizes hnRNAs, which are precursors to mRNAs. It also make most small nuclear RNAs (snRNAs Polymerase III Nucleoplasm Makes the precursor to 5SrRNA, the tRNAs and several other small cellular and viral RNAs.
  21. 21. Transcription Factors  Transcription factors are proteins that bind to DNA near the start of transcription of a gene, but they are not part of RNA polymerase molecule .  Transcription factors either inhibit or assist RNA polymerase in initiation and maintenance of transcription. 22
  22. 22. Regulatory elements Eukaryotic Promoter Conserved eukaryotic promoter elements Consensus sequence CAAT box GGCCAATCT TATA box TATAA GC box GGGCGG CAP site TAC 23 Eukaryotic Promoter lies adjacent to the gene, upstream to the transcription startpoint, serve as a recognition point that bind RNA polymerase (initiate transcription). There are several different types of promoter found in human genome, with different structure and different regulatory properties class/I/II/III.
  23. 23. Enhancers Enhancers are stretches of bases within DNA, about 50 to 150 base pairs in length; the activities of many promoters are greatly increased by enhancers which can exert their stimulatory actions over distances of several thousands base pairs. It serves to increase the efficiency of transcription, so increase the rate. It allow RNA polymerase to bind DNA till reach the promotor. 24
  24. 24. Enhancers bind to transcription factors by at Least 20 different proteins Form a complex change the configuration of the chromatin folding, bending or looping of DNA.
  25. 25. Preinitiation Complex  The general transcription factors combine with RNA polymerase to form a preinitiation complex that is competent to initiate transcription as soon as nucleotides are available.  The assembly of the preinitiation complex on each kind of eukaryotic promoter (class II promoters recognized by RNA polymerase II) begins with the binding of an assembly factor to the promoter. 27
  26. 26. 28 Source:
  27. 27. • The normal structure of the chromatin suppresses the gene activity, making the DNA relatively inaccessible to transcription factors, and thus active transcription complex can’t occur. • Thus chromatin remodeling is needed ( it is a change in chromatin conformation in which proteins of nucleosomes are released from DNA , allowing DNA to be accessible for TFs and RNA polymerase).
  28. 28. Inactive chromatin remodeled into active chromatin by 2 biochemical modifications: 1. Acetylation of histone proteins by histone acetyl transferases which loosen the association between DNA and histone. 2. Specialised protein complexes disrupt the nucleosome structure near the gene’s promoter site. This protein complex slides histone along DNA transfer the histone to other location on DNA molecule.
  29. 29. Active chromatin can be deactivated by 3 biochemical reactions: 1. Histone deacetylation ( catalysed by histone deacetylase). 2. Histone methylation ( catalysed by histone methyl transferases). 3. Methylation of some DNA nucleotides by DNA methyl transferases. (Chromatin subjected to these modifications tends to be transcriptionaly silent)
  30. 30. Phases of transcription 32
  31. 31. Initiation  The polymerase binding causes the unwinding of the DNA double helix which expose at least 12 bases on the template.  This is followed by initiation of RNA synthesis at this starting point. 33
  32. 32. Initiation  The RNA polymerase starts building the RNA chain; it assembles ribonucleotides triphosphates: ATP; GTP; CTP and UTP into a strand of RNA.  After the first nucleotide is in place, the polymerase joins a second nucleotide to the first, forming the initial phosphodiester bond in the RNA chain. 34
  33. 33. Elongation  RNA polymerase directs the sequential binding of riboncleotides to the growing RNA chain in the 5' - 3' direction.  Each ribonucleotide is inserted into the growing RNA strand following the rules of base pairing. This process is repeated utill the desired RNA length is synthesized…………………….. 35
  34. 34. Termination  Terminators at the end of genes; signal termination. These work in conjunction with RNA polymerase to loosen the association between RNA product and DNA template. The result is that the RNA dissociate from RNA polymerase and DNA and so stop transcription.  The product is immature RNA or pre mRNA (Primary transcript). 36
  35. 35. Product of transcription  The primary product of RNA transcription; the hnRNAs contain both intronic and exonic sequences.  These hnRNAs are processed in the nucleus to give mature mRNAs that are transported to the cytoplasm where to participate in protein synthesis. 37
  36. 36. RNA Processing (Pre-mRNA → mRNA)  Capping  Splicing  Addition of poly A tail 38
  37. 37. RNA Processing  Capping  The cap structure is added to the 5' of the newly transcribed mRNA precursor in the nucleus prior to processing and subsequent transport of the mRNA molecule to the cytoplasm.  Splicing: Step by step removal of pre mRNA and joining of remaining exons; it takes place on a special structure called spliceosomes. 39
  38. 38. RNA Processing  Addition of poly A tail:  Synthesis of the poly (A) tail involves cleavage of its 3' end and then the addition of about 40- 200 adenine residues to form a poly (A) tail. 40
  39. 39. 41
  40. 40. Alternative Splicing  Alternative splicing: is a very common phenomenon in higher eukaryotes. It is a way to get more than one protein product out of the same gene and a way to control gene expression in cells. 42
  41. 41. 43
  42. 42. The Genetic Code The sequence of codons in the mRNA defines the primary structure of the final protein. Three nucleotides in mRNA (a codon)specify one amino acid in a protein. 44
  43. 43. The Genetic Code  The triplet sequence of mRNA that specify certain amino acid.  There are only four letters to this code (A, G, C and U) that represent 43= 64 different combination of bases; 61 of them code for 20 amino acids (AA); the last three codon (UAG,UGA,UAA) don not code for amino acids; they are termination codons.  Degenerate  More than one triplet codon specify the same amino acid. 45
  44. 44. The Genetic Code 46
  45. 45. DNA & RNA Codon DNA Codon RNA Codon 47
  46. 46. Translation plan TRANSLATION Complete protein Polypeptide chain Ribosomes Stop codon Start codon
  47. 47. Translation 49
  48. 48. Translation  Translation is the process by which ribosomes read the genetic message in the mRNA and produce a protein product according to the message's instruction. 50
  49. 49. The protein synthesis occur in 3 phases  Accurate and efficient initiation occurs; the ribosomes binds to the mRNA, and the first amino acid attached to its tRNA.  Chain elongation, the ribosomes adds one amino acid at a time to the growing polypeptide chain.  Accurate and efficient termination, the ribosomes releases the mRNA and the polypeptide. 51
  50. 50. Initiation The initiation phase of protein synthesis requires over 10 eukaryotic Initiation Factors (eIFs): Factors are needed to recognize the cap at the 5'end of an mRNA and binding to the 40s ribosomal subunit. Binding the initiator Met-tRNAiMet (methionyl- tRNA) to the 40S small subunit of the ribosome. 52
  51. 51. Requirement for Translation Ribosomes tRNA mRNA  Amino acids Initiation factors Elongation factors Termination factors Aminoacyl tRNA synthetase enzymes: Energy source 53
  52. 52. Initiation Scanning to find the start codon by binding to the 5'cap of the mRNA and scanning downstream until they find the first AUG (initiation codon). The start codon must be located and positioned correctly in the P site of the ribosome, and the initiator tRNA must be positioned correctly in the same site. Once the mRNA and initiator tRNA are correctly bound, the 60S large subunit binds to form 80s initiation complex with a release of the eIF factors. 54
  53. 53. Elongation Transfer of proper aminoacyl-tRNA from cytoplasm to A-site of ribosome; Peptide bond formation; Peptidyl transferase forms a peptide bond between the amino acid in the P site, and the newly arrived aminoacyl tRNA in the A site. This lengthens the peptide by one amino acids. 55
  54. 54. Elongation Translocation; translocation of the new peptidyl t- RNA with its mRNA codon in the A site into the free P site occurs. Now the A site is free for another cycle of aminoacyl t-RNA codon recognition and elongation. Each translocation event moves mRNA, one codon length through the ribosomes. 56
  55. 55. Termination Translation termination requires specific protein factors identified as releasing factors, RFs in E. coli and eRFs in eukaryotes. The signals for termination are the same in both prokaryotes and eukaryotes. These signals are termination codons present in the mRNA. There are 3 termination codons, UAG, UAA and UGA. 57
  56. 56. Termination After multiple cycles of elongation and polymerization of specific amino acids into protein molecules, a nonsense codon = termination codon of mRNA appears in the A site. The is recognized as a terminal signal by eukaryotic releasing factors (eRF) which cause the release of the newly synthesized protein from the ribosomal complex. 58
  57. 57. Polysomes  Most mRNA are translated by more than one ribosome at a time; the result, a structure in which many ribosomes translate a mRNA in tandem, is called a polysomes. 59
  58. 58. Control of Gene Expression  Transcriptional  Posttranscriptional  Translational  Posttranslational 60
  59. 59. Control of Gene Expression 61
  60. 60. Control of gene expression Control of gene expression depends various factors including: Chromosomal activation or deactivation.  Control of initiation of transcription.  Processing of RNA (e.g. splicing).  Control of RNA transport.  Control of mRNA degradation.  Control of initiation of translation (only in eukaryotes).  Post-translational modifications. 62
  61. 61. Summery: Eukaryotic Gene Expression  Essentially all humans' genes contain introns. A notable exception is the histone genes which are intronless.  Eukaryote genes are not grouped in operons. Each eukaryote gene is transcribed separately, with separate transcriptional controls on each gene.  Eukaryotic mRNA is modified through RNA splicing.  Eukaryotic mRNA is generally monogenic (monocistronic); code for only one polypeptide. 63
  62. 62. Summery: Eukaryotic Gene Expression  Eukaryotic mRNA contain no Shine-Dalgarno sequence to show the ribosomes where to start translating. Instead, most eukaryotic mRNA have caps at their 5` end which directs initiation factors to bind and begin searching for an initiation codon.  Eukaryotes have a separate RNA polymerase for each type of RNA.  In eukaryotes, polysomes are found in the cytoplasm.  Eukaryotic protein synthesis initiation begins with methionine not N formyl- methionine. 64
  63. 63. Prokaryotic vs. Eukaryotic  Bacterial genetics are different.  Prokaryote genes are grouped in operons.  Prokaryotes have one type of RNA polymerase for all types of RNA,  mRNA is not modified  The existence of introns in prokaryotes is extremely rare. 65
  64. 64. Prokaryotic vs. Eukaryotic To initiate transcription in bacteria, sigma factors bind to RNA polymerases. RNA polymerases/ sigma factors complex can then bind to promoter about 40 deoxyribonucleotide bases prior to the coding region of the gene. In prokaryotes, the newly synthesized mRNA is polycistronic (polygenic) (code for more than one polypeptide chain). In prokaryotes, transcription of a gene and translation of the resulting mRNA occur simultaneously. So many polysomes are found associated with an active gene. 66
  65. 65. Gene Expression Analysis • Polymerase Chain Reaction • Quantitative PCR • Microarray • ……. • …… 67
  66. 66. Taq Polymerase • Thermus aquaticus DNA polymerase • thermophilic organism • enzymes resistant to high temperatures • 72-74o optimum PCR Requirements • heat-stable DNA polymerase • thermocycler • target DNA and primers
  67. 67. STEP TEMP TIME NOTES Denature 94-96 o 0.5-2 min longer:  denaturation, but  enzyme, template Annealing 15-25 o < Tm 0.5-2 min shorter:  specificity, but  yield Extension 72-75 o ~1 min (<kb) Taq processivity = 150 nucleotides/sec • mix DNA, primers, dNTPs, Taq, buffer, Mg2+ • program thermocycler for times and temps –denaturation –annealing –extension • 20-40 cycles • analyze amplified DNA (amplicons) PCR Protocol
  68. 68. Disadvantage of traditional PCR * Low sensitivity * Short dynamic range * Low resolution * Non-automated * Size-based discrimination only * Results are not expressed as numbers * Ethidium bromide staining is not very quantitative 1. Why Real-time PCR ?
  69. 69. Advantages of real-time PCR • amplification can be monitored real-time • wider dynamic range of up to 1010-fold • no post-PCR processing of products (No gel-based analysis at the end of the PCR reaction) • ultra-rapid cycling (30 minutes to 2 hours) • highly sequence-specific 1. Why Real-time PCR ?
  70. 70. 1.It requires expensive equipments and reagents 2.Due to its extremely high sensitivity, you may get high deviations of the same experiment, thus, the use of internal control genes is a recommended (in gene expression experiments) Disadvantages of real-time PCR 1. Why Real-time PCR ?
  71. 71. The QPCR Approach Chemistry l Use fluorescent dyes and probes l Establish a linear correlation between PCR product and fluorescence intensity Detection l Fluorescence detection to monitor amplification in real time Analysis l Software for analysis and estimation of template concentration 2- Theory of Real-time PCR ?
  72. 72. Concept of quantifications • RT-PCR is identical to a standard PCR except that the progress of RT-PCR is monitored by a detector at each cycle. • Each have used a kind of fluorescent marker which binds to DNA. • As the numbers of copies of genes increases; the reaction of fluorescence increases. • Quantifications is achieved by measuring the increase of fluorescence during the exponential phase of PCR.
  73. 73. qPCR detection-instrumention How are the PCR product (amplicon) detected in real time? • By combining a PCR thermal cycler with a fluorimeter • qPCR instruments are commonly configured to detect 2-5 different colors (or channels). • Multiple detection channels allow for quantifications of more than one target in one single tube (multiplex qPCR).
  74. 74. cDNA microarray schema From Duggan et al. Nature Genetics 21, 10 – 14 (1999) color code for relative expression
  75. 75. cDNA microarray raw data Yeast genome microarray. The actual size of the microarray is 18 mm by 18 mm. (DeRisi, Iyer & Brown, Science, 268: 680-687, 1997) • can be custom-made in the laboratory • always compares two samples • relatively cheap • up to about 20,000 mRNAs measured per array • probes about 50 to a few hundred nucleotides
  76. 76. Liver-enriched transcription factors • Liver-specific gene expression is controlled primarily at a transcriptional level. • Transcriptional regulatory elements of genes expressed in hepatocytes have identified several liver-enriched transcription factors (LETFs) which are key components of the differentiation process for the fully functional liver.
  77. 77. Glossary  Alleles are forms of the same gene with small differences in their sequence of DNA bases.  Alternative splicing: is a very common phenomenon in higher eukaryotes. It is a way to get more than one protein product out of the same gene and a way to control gene expression in cells.  Exon: a segment of a gene that is represented in the mature RNA product. Individual exons may contain coding DNAand/or noncoding DNA (untranslated sequences).  Bioinformatics I is the application of computer science and information technology to the field of biology and medicine  Introns (intervening sequence) (A noncoding DNA sequence ): Intervening stretches of DNA that separate exons.  Primary transcript: The initial production of gene transcription in the nucleus; an RNA containing copies of all exons and introns.  RNA gene or non-coding RNA gene: RNA molecule that is not translated into a protein. Noncoding RNA genes produce transcripts that exert their function without ever producing proteins. Non-coding RNA genes include transfer RNA (tRNA) and ribosomal RNA (rRNA), small RNAs such as snoRNAs, microRNAs, siRNAsand piRNAs and lastly long ncRNAs.  Enhancers and silencers: are DNA elements that stimulate or depress the transcription of associated genes; they rely on tissue specific binding proteins for their activities; sometimes a DNA elements can act either as an enhancer or silencer depending on what is bound to it.  Activators: Additional gene-specific transcription factors that can bind to enhancer and help in transcription activation.  Open reading frame (ORF): A reading frame that is uninterrupted by translation stop codon (reading frame that contains a start codon and the subsequent translated region, but no stop codon).  Directionality: in molecular biology, refers to the end-to-end chemical orientation of a single strand of nucleic acid. The chemical convention of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5' end and a 3' end ( "five prime end" and "three prime end"). The relative positions of structures along a strand of nucleic acid, including genes, transcription factors, and polymerases are usually noted as being either upstream (towards the 5' end) or downstream (towards the 3' end).  Reverse Transcription: Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA.  Pseudogenes. DNA sequences that closely resemble known genes but are nonfunctional.  More: 81