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DNA ... the genetic material.

Goals. Understand how DNA and the information it contains is… replicated, expressed, - altered by mutation. DNA ... the genetic material. D eoxyribo n ucleic A cid:

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DNA ... the genetic material.

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  1. Goals • Understand how DNA and the information it contains is… • replicated, • expressed, • - altered by mutation. DNA ...the genetic material. Deoxyribonucleic Acid: • a double stranded, helical nucleic acid molecule capable of replicating and determining* the inherited structure of a cell’s proteins.

  2. Replication transcription transcription Transcriptome translation translation Proteome Central Dogma(s) DNA Genome RNA Protein

  3. Genome Transcriptome Proteome Expanded Central Dogma • Genome... the dynamic complement of heritable genetic material, • Transcriptome... mRNA component in an individual, • complexity increases resulting from transcription control and transcription and post-transcription modifications, • Proteome... the protein component of an individual, • complexity increases due to translational efficiancy, post-translational modification, protein-protein interactions, etc.

  4. But, only ~20,000 genes? Complexity • 1021 stars (estimated) in the Universe, • astronomical, • If the human genome codes for ~100,000 proteins, all possible combinations of genes being off or on yields ~1030,000 discrete states, • per cell, • at any one moment over the span of a lifetime.

  5. Central Dogma genetics focus DNA transcription Replication RNA translation Protein

  6. Nucleic AcidsDNA/RNA …polymers consisting of monomers termed nucleotides, A, G, C, T/U • nucleotides: a molecule composed of, • a pentose sugar, • a phosphate group, • and an organic molecule called a nitrogenous base.

  7. Phosphodiester Backbones Mb long “Bases” Polynucleotides Phosphodiester BondsNote 5’- 3’Orientation Adenine Thymine Guanine Cytosine 3’ 5’

  8. Base Pairing A -- T complementary Base Pairing G -- C complementary Two Hydrogen Bonds Three Hydrogen Bonds Phosphodiester Backbones Bases with hydrogen bonds.

  9. Anti-Parallel ...DNA is a double stranded molecule and orients in an anti-parallel fashion, …orientation is in reference to the phosphodiester bonds. Complementary: strands aren’t identical, yet…????????

  10. Please study this slide.

  11. Strands can be separated. Complementary StrandsTemplates for Copying …single-stranded DNA can serve as a template for high-fidelity “duplication”, • makes DNA for growth, repair and hereditary purposes (Replication), • makes RNA for the synthesis of proteins (Transcription).

  12. -A -a 5’-AAAGGCTGATCA-3’ -B -b 3’-TTTCCGACTAGT-5’ Homologous Chromosomes Sister Chromatids DNA Replicationmeiosis I A- a- B- b- One Chromosome 3’-TTTCCGACTAGT-5’ 5’-AAAGGCTGATCA-3’ 3’-TTTCCGACTAGT-5’ 5’-AAAGGCTGATCA-3’

  13. DNA Replication • Template, • Enzymes, • Primer (to prime synthesis), • dNTPs: • d (deoxy), N (A,T,G,or C), • Energy.

  14. Enzyme #1 • Helicases: enzymes that unwind the DNA double helix for DNA replication… • Dna/A, Dna/B, Dna/C, • - proteins that bind to specific DNA sequences.

  15. Enzyme 2 Primase …Primase: provides a short, complementary strand of RNA that is required for DNA synthesis from a naked DNA template.

  16. Energy DNA polymerase III: the enzyme that adds complementary nucleotides to the backbone, based on the sequence of the single stranded template. 5’ --> 3’ synthesis. Enzyme 3

  17. DNA Synthesis test yourself

  18. Fidelity So FarMitosis …the initial synthesis produces errors at a rate of 1 in 105-8, …proof-reading during replication improves the error rate to 1 in 108-12, …this occurs at a rate of up to 1000 bp a second.

  19. Central Dogma genetics focus DNA transcription RNA translation Protein

  20. RNA Structure …single stranded, …can form base pairing with DNA, or RNA, …no simple regular secondary structure.

  21. mRNA, tRNA and rRNA …mRNA (messenger RNA): a type of RNA synthesized from DNA that specifies the primary structure for a protein, …tRNA (transfer RNA): an RNA molecule that acts as an ‘interpreter’ between nucleic language and protein language by picking up specific amino acids and recognizing the appropriate codons in the mRNA, …rRNA (ribosomal RNA): together with proteins, it forms the structure of ribosomes that coordinate the sequential coupling of tRNA molecules to the series of mRNA codons.

  22. Promoter Gene Expression…refers to cellular control of transcription, …non-transcribed DNA, a short distance from 5’end of a gene, …RNA polymerase is weakly attracted to DNA in general, but is strongly attracted to promoter sequences and associated molecules. 5’ DNA sequences and associated molecules … • direct when and where a gene is expressed, • influence the amount of expression, • strong promoter (high rate of transcription), • weak promoter (low rate of transcription). 5’ 3’ Structural Region (peptide sequence)

  23. Multiple Sequence Alignment E. coli Promoter Sequencesconsensus sequence alignment (from a MSA)

  24. Promoter Regions DNA sequence changes alter promoter function.

  25. cis-acting elements; DNA sequences that serve as attachments sites for the DNA-binding proteins that regulate the initiation of transcription. trans-acting elements; the DNA-binding proteins that regulate the initiation of transcription. Transcription Factor Terms Modulators of expression can act at great distances.

  26. Eukaryotic Initiation Apply the terms from the previous slide to the appropriate components on this figure. Please study this slide.

  27. Structural Regioncoding region …the portion of a gene that specifically codes for a protein. Terminator 5’ 3’ transcription unit RNA polymerase begins transcription here.

  28. Strands can be separated. Complementary StrandsTemplates for Copying …single-stranded DNA can serve as a template for high-fidelity “duplication”, • makes DNA for growth, repair and hereditary purposes (Replication), • makes RNA for the synthesis of proteins (Transcription).

  29. …from one strand of the double helix, DNA template strand is read 3’ to 5’, RNA strand ‘grows from 5’ to 3’, RNA Synthesis

  30. Coding strand Elongation Nucleotides are added to the 3’ end of the elongating RNA. • Template (DNA) and Promoter, • Nucleoside triphosphates (NTPs), • N: A,U,G,or C, • Enzymes (RNA polymerases), • Energy (phosphate bonds).

  31. Question: what does the coding strand sequence tell you about the mRNA sequence?

  32. hnRNA vs. mRNAheterogeneous nuclear vs. messenger • prokaryotic mRNA synthesis described so far requires little, or no further modification prior to translation into proteins, • eukaryotic transcripts requires extensive modifications.

  33. …lots of adenines added to the 3’ end. …modified guanine cap added to the 5’ end. Post Transcriptional Modification Ieukaryotes Occurs in the nucleus. Increases stability, may help transport and sorting.

  34. Exons code for peptides. Intervening sequences (introns), do not code for proteins. Post Transcriptional Modification IIeukaryotes Introns may alter expression. Differential splicing can alter the final protein’s structure and function. Provides “functional cassettes”, for evolutionary mixing and matching.

  35. Eukaryotic Intron Excision(sequence is important)

  36. Spliceosomesconfer sequence specificity ... small nuclear RNAs (snRNAs): • RNA molecules that act as catalysts in spliceosomes, …work in concert with > 100 proteins to facilitate intron identification and removal, • snRNPs: RNA/Protein structures.

  37. Psuedouradine (y) U1 and U2snRNAs U1 binds to the 5’ exon/intron junction. U2 binds to the adenosine region at the branch site. Think about the required specificity for intron identification in cells.

  38. …alternate splicing. Alternate mRNA Procesing

  39. …complicated biochemistry, lots of protein sub-units, • endonuclease, • polyadenylate polymerase, Polyadenylation…lots of adenines. AAUAA: consensus poly-A recognition site.

  40. ~20,000 genes --> ~100,000 proteins …alternate splicing. Alternate mRNA Processing …recognition of different poly-A sites.

  41. Complexity Calcitonin gene

  42. GeneticsIn the News “Devil Anse” Hatfield

  43. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM

  44. Translation …RNA is an intermediary in the transfer of information from DNA to the synthesis of protein, …how is that information organized?

  45. …involved in translation. mRNA, tRNA and rRNA …mRNA (messenger RNA): a type of RNA synthesized from DNA that specifies the primary structure for a protein, …tRNA (transfer RNA): an RNA molecule that acts as an ‘interpreter’ between nucleic language and protein language by picking up specific amino acids by recognizing the appropriate codon in the mRNA, …rRNA (ribosomal RNA): together with proteins, it forms the structure of ribosomes that coordinate the sequential coupling of tRNA molecules from the series of mRNA codons.

  46. tRNA secondary structure ‘reveals’ a three base pair structure on one end, The ‘Ends’ to the Means Specific anti-codons for specific amino acids. “anti” = complementary

  47. Ribosomes …a supramolecular complex of rRNA and proteins, approximately 18 - 22 nm in diameter, …the site of protein synthesis,

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