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Understanding DNA Replication: Key Concepts and Processes

Explore the intricate process of DNA replication in E. coli genome, including genes, chromosomes, and genetic variation. Learn about DNA polymerase, leading and lagging strands, and key enzymes like helicase and primase. Discover how cells ensure accurate DNA synthesis and division.

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Understanding DNA Replication: Key Concepts and Processes

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  1. Chromosomes The E. Coli genome includes approximately 4,000 genes • Chromosomes • Strands of DNA that contain all of the genes an organism needs to survive and reproduce • Genes • Segments of DNA that specify how to build a protein • genes may specify more than one protein in eukaryotes • Chromosome maps are used to show the locus (location) of genes on a chromosome

  2. Chromosomes • Genetic Variation • Phenotypic variation among organisms is due to genotypic variation (differences in the sequence of their DNA bases) • Differences exist between species and within a species • Different genes (genomes)  different proteins (proteomes) • Different versions of the same gene (alleles) • Differences in gene expression (epigenetics)

  3. DNA Replication • Cell Division (mitosis) • Cells must copy their chromosomes (DNA synthesis) before they divide so that each daughter cell will have a copy • A region of the chromosome remains uncopied (centromere) in order to hold the sister chromatids together • Keeps chromatids organized to help make sure each daughter cell gets exactly one copy • Nondisjunction is when sister chromatids do not assort correctly and one cell ends up with both copies while the other cell ends up with none

  4. DNA Replication A G C T G T C G A C A G C T G T C G A C A G C T G T C G A C A G C T G T C G A C A G C T G T C G A C • DNA Synthesis • The DNA bases on each strand act as a template to synthesize a complementary strand • Recall that Adenine (A) pairs with thymine (T)and guanine (G) pairs with cytosine (C) • The process is semiconservative because each new double-stranded DNA contains one old strand (template) and one newly-synthesized complementary strand

  5. DNA Replication • DNA Polymerase • Enzyme that catalyzes the covalent bond between the phosphate of one nucleotide and the deoxyribose (sugar) of the next nucleotide DNA Polymerization

  6. DNA Replication • 3’ end has a free deoxyribose • 5’ end has a free phosphate • DNA polymerase: • can only build the new strand in the 5’ to 3’ direction • Thus scans the template strand in 3’ to 5’ direction

  7. DNA Replication DNA polymerase • Initiation • Primase (a type of RNA polymerase)builds an RNAprimer(5-10 ribonucleotides long) • DNA polymerase attaches onto the 3’ end of the RNAprimer

  8. DNA Replication • Elongation • DNA polymerase uses each strand as a template in the 3’ to 5’ direction to build a complementary strand in the 5’ to 3’ direction DNA polymerase

  9. DNA Replication • Elongation • DNA polymerase uses each strand as a template in the 3’ to 5’ direction to build a complementary strand in the 5’ to 3’ direction • results in a leading strand and a lagging strand

  10. DNA Replication • Leading Strand • Topisomerase unwinds DNA and then Helicase breaks H-bonds • DNA primase creates a single RNA primer to start the replication • DNA polymerase slides along the leading strand in the 3’ to 5’ direction synthesizing the matching strand in the 5’ to 3’ direction • The RNA primer is degraded by RNase H and replaced with DNA nucleotides by DNA polymerase, and then DNA ligase connects the fragment at the start of the new strand to the end of the new strand (in circular chromosomes)

  11. DNA Replication • Lagging Strand • Topisomerase unwinds DNA and then Helicase breaks H-bonds • DNA primase creates RNA primers in spaced intervals • DNA polymerase slides along the leading strand in the 3’ to 5’ direction synthesizing the matching Okazaki fragments in the 5’ to 3’ direction • The RNA primers are degraded by RNase H and replaced with DNA nucleotides by DNA polymerase • DNA ligase connects the Okazaki fragments to one another (covalently bonds the phosphate in one nucleotide to the deoxyribose of the adjacent nucleotide)

  12. DNA Replication Topoisomerase - unwinds DNA Helicase – enzyme that breaks H-bonds DNA Polymerase – enzyme that catalyzes connection of nucleotides to form complementary DNA strand in 5’ to 3’ direction (reads template in 3’ to 5’ direction) Leading Strand – transcribed continuously in 5’ to 3’ direction Lagging Strand – transcribed in segments in 5’ to 3’ direction (Okazaki fragments) DNA Primase – enzyme that catalyzes formation of RNA starting segment (RNA primer) DNA Ligase – enzyme that catalyzes connection of two Okazaki fragments

  13. Web Resources • DNA Replication (synthesis) • http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter11/animation_quiz_2.html • http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html • http://www.biostudio.com/d_%20DNA%20Replication%20Coordination%20Leading%20Lagging%20Strand%20Synthesis.htm • http://www.biostudio.com/d_%20DNA%20Replication%20Nucleotide%20Polymerization.htm • http://www.dnalc.org/resources/3d/DNAReplicationBasic_w_FX.html (download this video file from the website to view it without interruptions) • http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/dna-rna2.swf • http://www.bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplication.swf

  14. Protein Synthesis Nucleotide sequence of His gene • DNA provides the instructions for how to build proteins • Each gene dictates how to build a single protein in prokaryotes • The sequence of nucleotides (AGCT) in DNA dictate the order of amino acids that make up a protein

  15. Protein Synthesis Nucleotide sequence of His gene Amino acid sequence of His protein • DNA provides the instructions for how to build proteins • Each gene dictates how to build a single protein in prokaryotes • The sequence of nucleotides (AGCT) in DNA dictate the order of amino acids that make up a protein

  16. Protein Synthesis 1 2 mRNA (messenger RNA) copy of a gene is synthesized • Cytoplasm of prokaryotes • Nucleus of eukaryotes mRNA is used by ribosome to build protein (Ribosomes attach to the mRNA and use its sequence of nucleotides to determine the order of amino acids in the protein) • Cytoplasm of prokaryotes and eukaryotes • Some proteins feed directly into rough ER in eukaryotes • Protein synthesis occurs in two primary steps

  17. Protein Synthesis 1) INITIATION (eukaryotes) • TranscriptionInitiation • RNA polymerase binds to a region on DNA known as the promoter, which signals the start of a gene • Promoters are specific to genes • RNA polymerase does not need a primer • Transcription factors assemble at the promoter forming a transcription initiation complex– activator proteins help stabilize the complex • Gene expression can be regulated (turned on/off or up/down) by controlling the amount of each transcription factor

  18. Protein Synthesis 1) INITIATION • TranscriptionElongation • RNA polymerase unwinds the DNA and breaks the H-bonds between the bases of the two strands, separating them from one another • Base pairing occurs between incoming RNA nucleotides and the DNA nucleotides of the gene (template) • recall RNA uses uracil instead of thymine AGTCAT UCA GUA

  19. Protein Synthesis • TranscriptionElongation • RNA polymerase unwinds the DNA and breaks the H-bonds between the bases of the two strands, separating them from one another. • Base pairing occurs between incoming RNA nucleotides and the DNA nucleotides of the gene (template) • recall RNA uses uracil instead of thymine • RNA polymerase catalyzes bond to form between ribose of 3’ nucleotide of mRNA and phosphate of incoming RNA nucleotide 5’ 3’ + ATP 5’ 3’ + ADP

  20. Protein Synthesis • TranscriptionElongation The gene occurs on only one of the DNA strands; each strand possesses a separate set of genes

  21. Protein Synthesis 1) INITIATION • TranscriptionTermination • A region on DNA known as the terminator signals the stop of a gene • RNA polymerase disengages the mRNA and the DNA

  22. Protein Synthesis • Exons are “coding” regions • Introns are removed • different combinations of exons form different mRNA resulting in multiple proteins from the same gene • Humans have 30,000 genes but are capable of producing 100,000 proteins • Alternative Splicing (eukaryotes only)

  23. Web Resources • Transcription • http://www.biostudio.com/d_%20Transcription.htm • http://www.youtube.com/watch?v=WsofH466lqk • http://www.dnalc.org/resources/3d/TranscriptionBasic_withFX.html • Alternative Splicing • http://www.youtube.com/watch?v=FVuAwBGw_pQ&feature=related

  24. Protein Synthesis Transcription tRNA synthesis 1 mRNA mRNA copy of a gene is synthesized • Cytoplasm of prokaryotes • Nucleus of eukaryotes Translation 2 mRNA mRNA is used by ribosome to build protein (Ribosomes attach to the mRNA and use its sequence of nucleotides to determine the order of amino acids in the protein) • Cytoplasm of prokaryotes and eukaryotes • Some proteins feed directly into rough ER in eukaryotes

  25. Protein Synthesis Transcription tRNA synthesis mRNA Translation • Translation • Every three mRNA nucleotides (codon) specify an amino acid

  26. Protein Synthesis • Translation • tRNA have an anticodon region that specifically binds to its codon

  27. Protein Synthesis Transcription tRNA synthesis mRNA Translation • Translation • Each tRNA carries a specific amino acid

  28. Protein Synthesis Transcription tRNA synthesis mRNA Translation Aminoacyl tRNA synthetases attach amino acids to their specific tRNA

  29. Protein Synthesis Transcription tRNA synthesis mRNA Translation • TranslationInitiation • Start codon signals where the gene begins (at 5’ end of mRNA) 5’ 3’ AUGGACAUUGAACCG… start codon

  30. Protein Synthesis Large ribosomal subunit Ribosome Small ribosomal subunit • TranslationInitiation • Start codon signals where the gene begins (at 5’ end of mRNA) • Ribosome binding site (Shine Dalgarno sequence) upstream from the start codon binds to small ribosomal subunit • then this complex recruits the large ribosomal subunit Small ribosomal subunit

  31. Protein Synthesis • TranslationScanning • The ribosome moves in 5’ to 3’ direction “reading” the mRNA and assembling amino acids into the correct protein large ribosome subunit small ribosome subunit

  32. Protein Synthesis • TranslationScanning • The ribosome moves in 5’ to 3’ direction “reading” the mRNA and assembling amino acids into the correct protein

  33. Protein Synthesis • TranslationTermination • Ribosome disengages from the mRNA when it encounters a stop codon

  34. Web Resources • Translation • Eukaryotic: http://www.youtube.com/watch?v=5bLEDd-PSTQ&feature=related • Prokaryotic: http://www.biostudio.com/d_%20Protein%20Synthesis%20Prokaryotic.htm • http://www.biostudio.com/d_%20Peptide%20Bond%20Formation.htm • http://www.johnkyrk.com/DNAtranslation.html • http://www.dnalc.org/resources/3d/TranslationBasic_withFX0.html • http://www.dnalc.org/resources/3d/TranslationAdvanced.html

  35. Practice Question Translate the following mRNA sequence AGCUACCAUACGCACCCGAGUUCUUCAAGC

  36. Practice Question Translate the following mRNA sequence AGCUACCAUACGCACCCGAGUUCUUCAAGC Serine – Tyrosine – Histidine – Threonine – Histidine – Proline – Serine – Serine – Serine - Serine

  37. Practice Question Translate the following mRNA sequence AGCUACCAUACGCACCCGAGUUCUUCAAGC Serine – Tyrosine – Histidine – Threonine – Histidine – Proline – Serine – Serine – Serine - Serine Ser – Tyr – His – Thr – His – Pro – Ser – Ser – Ser - Ser

  38. Practice Question Translate the following mRNA sequence AGCUACCAUACGCACCCGAGUUCUUCAAGC Serine – Tyrosine – Histidine – Threonine – Histidine – Proline – Serine – Serine – Serine - Serine Ser – Tyr – His – Thr – His – Pro – Ser – Ser – Ser - Ser S – Y –H– T – H – P – S – S – S - S

  39. Protein Synthesis mRNAs DNA Translation • Multiple RNA polymerases can engage a gene at one time • Multiple ribosomes can engage a single mRNA at one time Transcription

  40. Protein Synthesis • Eukaryotes: transcription occurs in the nucleus and translation occurs in the cytoplasm • Prokaryotes: Transcription and translation occur simultaneously in the cytoplasm

  41. RNA • There are four main types of RNA: • mRNA- RNA copy of a gene used as a template for protein synthesis • rRNA- part of structure of ribosomes • tRNA- amino acid carrier that matches to mRNA codon • snRNA- found in nucleus where they have several important jobs

  42. Practice Questions • Why is DNA synthesis said to be “semiconservative”? • What role do DNA polymerase, DNA primase (a type of RNA polymerase), helicase, topoisomerase, RNase H, and ligase play in DNA replication? • What is the difference between how the leading strand and lagging strand are copied during DNA replication? Why do they have to be synthesized differently in this fashion? • What would happen if insufficient RNase H were produced by a cell? What if insufficient ligase were produced by a cell? • What are four key differences between DNA polymerase and RNA polymerase? (“they are difference molecules” doesn’t count as one!) • Compare and contrast codons and anticodons? • What is alternative splicing? Why is it necessary in eukaryotes? • During translation, what amino acid sequence would the following mRNA segment be converted into: AUGGACAUUGAACCG? • How come there are only 20 amino acids when there are 64 different codons? • How come prokaryotes can both transcribe and translate a gene at the same time, but eukaryotes cannot?

  43. Web Resources • Transcription • http://www.biostudio.com/d_%20Transcription.htm • http://www.youtube.com/watch?v=WsofH466lqk • http://www.dnalc.org/resources/3d/TranscriptionBasic_withFX.html • Alternative Splicing • http://www.youtube.com/watch?v=FVuAwBGw_pQ&feature=related • Translation • Eukaryotic: http://www.youtube.com/watch?v=5bLEDd-PSTQ&feature=related • Prokaryotic: http://www.biostudio.com/d_%20Protein%20Synthesis%20Prokaryotic.htm • http://www.biostudio.com/d_%20Peptide%20Bond%20Formation.htm • http://www.johnkyrk.com/DNAtranslation.html • http://www.dnalc.org/resources/3d/TranslationBasic_withFX0.html • http://www.dnalc.org/resources/3d/TranslationAdvanced.html

  44. Web Resources Insulin Example of Protein Synthesis http://www.biotopics.co.uk/as/insulinproteinstructure.html Hemoglobin Example of Protein Synthesis http://www.biotopics.co.uk/as/insulinproteinstructure.html Collagen Example of Protein Synthesis http://www.biotopics.co.uk/JmolApplet/collagen.html

  45. Images • http://www.kscience.co.uk/as/module1/pictures/bacteria.jpg • http://www.biologie.uni-hamburg.de/b-online/library/onlinebio/14_1.jpg • http://pharmamotion.com.ar/wp-content/uploads/2009/12/nrti_mechanism_action_antiretrovirals.jpg • http://biology200.gsu.edu/houghton/4564%20%2704/figures/lecture%204/AAAreverse.jpg • http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/2d8x/traces.jpg • http://www.ncbi.nlm.nih.gov • http://xarquon.jcu.cz/edu/uvod/09nucleus/092function/images/activation3.jpg • http://www.ncbi.nlm.nih.gov • http://bass.bio.uci.edu/~hudel/bs99a/lecture23/lecture4_4.html • http://selfhpvdna.diagcorlab.com/images/images/CervicalCancer.jpg

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