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How to Study DNA

How to Study DNA. Genetic material Expression product. What is gene expression?. The activation of a gene that results in a protein. Biological processes, such as transcription, and in case of proteins, also translation, that yield a gene product.

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How to Study DNA

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  1. How to Study DNA • Genetic material • Expression product

  2. What is gene expression? • The activation of a gene that results in a protein. • Biological processes, such as transcription, and in case of proteins, also translation, that yield a gene product. • A gene is expressed when its biological product is present and active. • Gene expression is regulated at multiple levels.

  3. Expression of Genetic Information Production of proteins requires two steps: • Transcription involves an enzyme (RNA polymerase) making an RNA copy of part of one DNA strand. There are four main classes of RNA: i. Messenger RNAs (mRNA), which specify the amino acid sequence of a protein by using codons of the genetic code. ii. Transfer RNAs (tRNA). iii. Ribosomal RNAs (rRNA). iv. Small nuclear RNAs (snRNA), found only in eukaryotes. • Translation converts the information in mRNA into the amino acid sequence of a protein using ribosomes, large complexes of rRNAs and proteins.

  4. Expression of Genetic Information • Only some of the genes in a cell are active at any given time, and activity also varies by tissue type and developmental stage. • Regulation of gene expression is not completely understood, but it has been shown to involve an array of controlling signals. a. Jacob and Monod (1961) proposed the operon model to explain prokaryotic gene regulation, showing that a genetic switch is used to control production of the enzymes needed to metabolize lactose. Similar systems control many genes in bacteria and their viruses. b. Genetic switches used in eukaryotes are different and more complex, with much remaining to be learned about their function.

  5. Steps of gene expression • Transcription – DNA is read to make a mRNA in the nucleus of our cells • Translation – Reading the mRNA to make a protein in the cytoplasm

  6. Three (3) regulatory elements of transcription Structural genes: DNA that code for a specific polypeptide (protein) Promoter : DNA segment that recognizes RNA polymerase Operator : Element that serves as a binding site for an inhibitor protein (modulator) that controls transcription

  7. Promoter Region on DNA • Upstream from transcription start site • Initial binding site of RNA polymerase and initiation factors (IFs) • Promoter recognition: a prerequisite for initiation Prokaryotic promoter regions -10 site: “TATA” box -35 site = TTGACA

  8. Promoter Region on DNA

  9. Pol II Eukaryotic Promoter Elements Exon Intron Exon GC box ~200 bp CCAAT box ~100 bp TATA box ~30 bp Gene Transcription start site (TSS)

  10. Pol II Eukaryotic Promoter Elements • Cap Region/Signal • n C A G T n G • TATA box (~ 25 bp upstream) • T A T A A A n G C C C • CCAAT box (~100 bp upstream) • T A G C C A A T G • GC box (~200 bp upstream) • A T A G G C G nGA

  11. General modulators of transcription • Modulators: (1) specificity factors, (2) repressors, (3) activators • Specificity factors: Alter the specificity of RNA polymerase s70 s32 Standard promoter Heat shock promoter Housekeeping gene Heat shock gene

  12. Modulators of transcription 2. Repressors: • mediate negative gene regulation • may impede access of RNA polymerase to the promoter • actively block transcription • bind to specific “operator” sequences (repressor binding sites) • Repressor binding is modulated by specific effectors Effector (e.g. endproduct) Repressor Operator Coding sequence Promoter

  13. Negative regulation Repressor Effector Example: lac operon RESULT: Transcription occurs when the gene is derepressed

  14. Negative regulation Repressor Effector (= co-repressor) Example: pur-repressor in E. coli; regulates transcription of genes involved in nucleotide metabolism

  15. Modulators of transcription 3. Activators: • mediate positive gene regulation • bind to specific regulatory DNA sequences (e.g. enhancers) • enhance the RNA polymerase -promoter interaction and actively stimulate transcription • common in eukaryotes RNA pol. Activator promoter Coding sequence

  16. Positive regulation Activator RNA polymerase

  17. Positive regulation Activator Effector RNA polymerase

  18. Prokaryotic gene organization Prokaryotic transcriptional regulatory regions (promoters and operators) lie close to the transcription start site Functionally related genes are frequently located near each other These “operons” are transcribed into a single mRNA with internal translation initiation sites

  19. Ribosome, tRNAs, Protein Factors Translation Prokaryotic Gene Expression Expression mainly by controlling transcription Promoter Cistron1 Cistron2 CistronN Terminator Transcription RNA Polymerase mRNA 5’ 3’ 1 2 N N N C N C C 1 2 3 Polypeptides

  20. Operons • Genes that work together are located together • A promoter plus a set of adjacent genes whose gene products function together. • They are controlled as a unit • They usually contain 2 –6 genes (up to 20 genes) • These genes are transcribed as a polycistronic transcript. • It is relatively common in prokaryotes • It is rare in eukaryotes

  21. Operon System

  22. Pi I P Q1 Z Y A Q3 Q2 The lactose (lac) operon • Contains several elements • lacZ gene = β-galactosidase • lacY gene = galactosidase permease • lacA gene = thiogalactoside transacetylase • lacI gene = lac repressor • Pi = promoter for the lacI gene • P = promoter for lac-operon • Q1 = main operator • Q2 and Q3 = secondary operator sites (pseudo-operators)

  23. LacZ LacY LacA • Inducer molecules→ Allolactose: • natural inducer, degradable IPTG • (Isopropylthiogalactoside) • - synthetic inducer, not metabolized lacI repressor Pi Pi I I P P Q1 Q1 Z Z Y Y A A Q3 Q3 Q2 Q2 Regulation of the lac operon

  24. The lac operon: model for gene expression • Includes three protein synthesis coding region--sometimes called "genes" as well as region of chromosome that controls transcription of genes • Genes for proteins involved in the catabolism or breakdown of lactose • When lactose is absent, no transcription of gene since no need for these proteins • When lactose is present, transcription of genes takes place so proteins are available to catalyze breakdown of lactose

  25. Eukaryotic gene

  26. Eukaryotic gene Expression 1.Transcripts begin and end beyond the coding region 2.The primary transcript is processed by: 5’ capping 3’ formation / polyA splicing 3.Mature transcripts are transported to the cytoplasm for translation

  27. Regulation of gene expression Promoter Gene (red) with an intron (green) Plasmid single copy vs. multicopy plasmids 1. DNA replication 2.Transcription Primary transcript mRNA degradation 3. Posttranscriptional processing Mature mRNA 4. Translation inactive protein Protein degradation 5. Posttranslational processing active protein

  28. Regulation of gene expression • Gene expression is regulated—not all genes are constantly active and having their protein produced • The regulation or feedback on gene expression is how the cell’s metabolism is controlled. • This regulation can happen in different ways: • 1. Transcriptional control (in nucleus): • e.g. chromatin density and transcription factors • 2. Posttranscriptional control (nucleus) • e.g. mRNA processing • 3. Translational control (cytoplasm) • e.g. Differential ability of mRNA to bind ribosomes • 4. Posttranslational control (cytoplasm) • e.g. changes to the protein to make it functional • When regulation of gene expression goes wrong—cancer!

  29. Transcription

  30. Eukaryotic gene expression

  31. Condition 2 “turned off” “turned on” 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 25 26 19 20 21 22 23 24 induced gene repressed gene constitutively expressed gene inducible/ repressible genes Gene regulation of the transcription Condition 1 “turned off” “turned on” Chr. I Chr. II Chr. III

  32. Condition 4 upregulated gene expression down regulated gene expression Gene regulation Condition 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 25 26 19 20 21 22 23 24 constitutively expressed gene

  33. Definitions • Constitutively expressed genes Genes that are actively transcribed (and translated) under all experimental conditions, at essentially all developmental stages, or in virtually all cells. • Inducible genes Genes that are transcribed and translated at higher levels in response to an inducing factor • Repressible genes Genes whose transcription and translation decreases in response to a repressing signal • Housekeeping genes • genes for enzymes of central metabolic pathways (e.g. TCA cycle) • these genes are constitutively expressed • the level of gene expression may vary

  34. Post-Transcriptional Modification in Eukaryotes • Primary transcriptformed first • Then processed (3 steps) to form mature mRNA • Then transported to cytoplasm Step 1: 7- methyl-guanosine “5’-cap” added to 5’ end Step 2: introns spliced out; exons link up Step 3: Poly-A tail added to 3’ end mature mRNA 5’-cap- exons -3’ PolyA tail

  35. Intron Splicing in Eukaryotes • Exons: coding regions • Introns :noncoding regions • Introns are removed by “splicing” AG at 3’ end of intron GU at 5’ end of intron

  36. Splicesomes Roles in Splicing out Intron RNA splicing occurs in small nuclear ribonucleoprotein particles (snRNPS) in spliceosomes

  37. Splicesomes Roles in Splicing out Intron • 5’ exon then moves to the 3’ splice acceptor site where a second cut is made by the spliceosome • Exon termini are joined and sealed 1 2 2 1 1 2

  38. Translation • Three parts: 1. Initiation: start codon (AUG) 2. Elongation: 3. Termination: stop codon (UAG)

  39. mRNA A U G C U A C U U C G Translation Large subunit P Site A Site Small subunit

  40. aa2 aa1 2-tRNA 1-tRNA G A U U A C Initiation anticodon A U G C U A C U U C G A hydrogen bonds codon mRNA

  41. aa3 3-tRNA G A A peptide bond aa1 aa2 1-tRNA 2-tRNA anticodon U A C G A U A U G C U A C U U C G A hydrogen bonds codon mRNA

  42. aa3 3-tRNA G A A aa1 peptide bond aa2 1-tRNA U A C (leaves) 2-tRNA G A U A U G C U A C U U C G A mRNA Ribosomes move over one codon

  43. aa4 4-tRNA G C U peptide bonds aa1 aa2 aa3 2-tRNA 3-tRNA G A U G A A A U G C U A C U U C G A A C U mRNA

  44. aa4 4-tRNA G C U peptide bonds aa1 aa2 aa3 2-tRNA G A U (leaves) 3-tRNA G A A A U G C U A C U U C G A A C U mRNA Ribosomes move over one codon

  45. aa5 5-tRNA U G A peptide bonds aa1 aa2 aa4 aa3 3-tRNA 4-tRNA G A A G C U G C U A C U U C G A A C U mRNA

  46. aa5 5-tRNA U G A peptide bonds aa1 aa2 aa3 aa4 3-tRNA G A A 4-tRNA G C U G C U A C U U C G A A C U mRNA Ribosomes move over one codon

  47. aa5 aa4 Termination aa199 aa200 aa3 primary structure of a protein aa2 aa1 terminator or stop codon 200-tRNA A C U C A U G U U U A G mRNA

  48. Ribosome Amino Acids forming Peptide chain P Site A Site E Site tRNA anti-codon AUG GGA codon Translation Met His Tyr Val Pro 3’ CAU UAC GUA CCU 5’ mRNA strand

  49. Translation The difference • Eukaryotic and prokaryotic translation can react differently to certain antibiotics Puromycin an analog tRNA and a general inhibitor of protein synthesis  Cycloheximide only inhibits protein synthesis by eukaryotic ribosomes  Chloramphenicol, Tetracycline, Streptomycin inhibit protein synthesis by prokaryotic ribosome

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