1. Genes and Protein Synthesis Chapter 7

2. One Gene-One Polypeptide Hypothesis DNA contains all of our hereditary information Genes are located in our DNA ~25,000 genes in our DNA (46 chromosomes) Each Gene codes for a specific polypeptide

3. Main Idea Central Dogma – Francis Crick (1956)

4. Overall Process Transcription – DNA to RNA Translation – Assembly of amino acids into polypeptide – Using RNA DNA molecule Gene 1 Gene 2 Gene 3 DNA strand TRANSCRIPTION RNA Polypeptide TRANSLATION Codon Amino acid

5. Key Terms RNA transcription – Initiation, Elongation, Termination TATA box Introns, Exons mRNA, tRNA, rRNA Translation Ribosome Codon Amino Acids Polypeptide

6. DNARNA Double strandedSingle stranded Adenine pairs with ThymineAdenine pairs with Uracil Guanine pairs with Cytosine Deoxyribose sugarRibose sugar

7. DNA to Protein Protein is made of amino acid sequences 20 amino acids How does DNA code for amino acid? DNA molecule Gene 1 Gene 2 Gene 3 DNA strand TRANSCRIPTION RNA Polypeptide TRANSLATION Codon Amino acid

8. Genetic Code Codon – Three letter code – 5’ to 3’ order – Start codon – Stop codon AA are represented by more than one codon 61 codons that specify AA

9. Amino acids Abbreviated – Three letters

10. Transcription DNA to RNA Occurs in nucleus Three process – Initiation – Elongation – Termination RNA polymerase DNA of gene Promoter DNA Terminator DNA Initiation Elongation Termination Growing RNA RNA polymerase Completed RNA

11. Initiation RNA polymerase binds to DNA Binds at promoter region – TATA box RNA polymerase unwinds DNA Transcription unit – Part of gene that is transcribed Transcription factors bind to specific regions of promoter Provide a substrate for RNA polymerase to bind beginning transcription Forms transcription initiation complex

12. Elongation RNA molecule is built – RNA polymerase Primer not needed 5’ to 3’ direction Template strand is copied – 3’ to 5’ DNA Coding strand – DNA strand that is not copied Produces mRNA – Messenger RNA DNA double helix reforms

13. Termination RNA polymerase recognizes a termination sequence – AAAAAAA (polyadenylation) Nuclear proteins bind to string of UUUUUU on RNA mRNA molecule releases from template strand

15. Post-Transcriptional Modifications Pre-mRNA undergoes modifications before it leaves the nucleus Poly(A) tail – Poly-A polymerase – Protects from RNA digesting enzymes in cytosol 5’ cap – 7 G’s – Initial attachment site for mRNA’s to ribosomes Removal of introns

16. Splicing the pre-mRNA DNA comprised of – Exons sequence of DNA or RNA that codes for a gene – Introns non-coding sequence of DNA or RNA Spliceosome – Enzyme that removes introns from mRNA

17. Splicing Process Spliceosome contains a handful of small ribonucleoproteins – snRNP’s (snurps) snRNP’s bind to specific regions on introns

18. Alternative Splicing Increases number and variety of proteins encoded by a single gene ~25,000 genes produce ~100,000 proteins

19. Translation mRNA to protein Ribosomes read codons tRNA assists ribosome to assemble amino acids into polypeptide chain Takes place in cytoplasm

20. tRNA Contains – triplet anticodon – amino acid attachment site Are there 61 tRNA’s to read 61 codons?

21. tRNA: Wobble Hypothesis First two nucleotides of codon for a specific AA is always precise Flexibility with third nucleotide Aminoacylation – process of adding an AA to a tRNA – Forming aminoacyl- tRNA molecule – Catalyzed by 20 different aminoacyl- tRNA synthetase enzymes

22. Ribosomes Translate mRNA chains into amino acids Made up of two different sized parts – Ribosomal subunits (rRNA) Ribosomes bring together mRNA with aminoacyl- tRNAs Three sites – A site - aminoacyl – P site – peptidyl – E site - exit

23. 1Codon recognition Amino acid Anticodon A site P site Polypeptide 2 Peptide bond formation 3 Translocation New peptide bond mRNA movement mRNA Stop codon Translation process Three stages – Initiation – Elongation – Termination

24. Initiation Ribosomal subunits associate with mRNA Met-tRNA (methionine) – Forms complex with ribosomal subunits Complex binds to 5’cap and scans for start codon (AUG) (scanning) Large ribosomal subunit binds to complete ribosome Met-tRNA is in P-site  Reading frame is established to correctly read codons

25. Elongation Amino acids are added to grow a polypeptide chain A, P, and E sites operate 4 Steps

26. Termination A site arrives at a stop codon on mRNA – UAA, UAG, UGA Protein release factor binds to A site releasing polypeptide chain Ribosomal subunits, tRNA release and detach from mRNA

27. b a Red object = ? What molecules are present in this photo?

28. Review What is a gene? Where is it located? What is the main function of a gene? Do we need our genes “on” all the time? How do we turn genes “on” or “off”?

29. Regulating Gene Expression Proteins are not required by all cells at all times Regulated Eukaryotes – 4 ways – Transcriptional (as mRNA is being synthesized) – Post-transcriptional (as mRNA is being processed) – Translational (as proteins are made) – Post-translational (after protein has been made)

30. Transcriptional regulation Most common DNA wrapped around histones keep gene promoters inactive Activator molecule is used (2 ways) – Signals a protein remodelling complex which loosen the histones exposing promoter – Signals an enzyme that adds an acetyl group to histones exposing promoter region

31. Transcriptional regulation Methylation – Methyl groups are added to the cytosine bases in the promoter of a gene (transcription initiation complex) – Inhibits transcription – silencing – Genes are placed “on hold” until they are needed – E.g. hemoglobin

32. Post transcriptional regulation Pre-mRNA processing – Alternative splicing Rate of mRNA degradation – Masking proteins used to degrade mRNA – Translation does not occur Embryonic development Hormones – Casein – milk protein in mammary gland – When casein is needed, prolactin is produced extending lifespan of casein mRNA

33. Translational regulation Occurs during protein synthesis by a ribosome Changes in length of poly(A) tail – Enzymes add or delete adenines – Increases or decreases time required to translate mRNA into protein – Environmental cues

34. Post-Translational Regulation Processing – Removes sections of protein to make it active – Cell regulates this process (hormones) Chemical modification – Chemical groups are added or deleted – Puts the protein “on hold” Degradation – Proteins tagged with ubiquitin are degraded – Amino acids are recycled for protein synthesis

35. Cancer Lack regulatory mechanisms Mutations in genetic code (mutagens) – Probability increases over lifetime – Radiation, smoking, chemicals Mutations are passed on to daughter cells – Can lead to a mass of undifferentiated cells (tumor) – Benign and malignant Oncogenes – Mutated genes that once served to stimulate cell growth – Cause undifferentiated cell division

36. Genetic Mutations Positive and negative – Natural selection – evolution – Cancer –death Small-Scale – single base pair – Point mutations Substitution, insertion/deletion, inversion Large-Scale – multiple base pairs

37. Small-Scale Mutations Four groups – Missense, nonsense, silent, frameshift Lactose, sickle cell anemia – SNPs – single nucleotide polymorphisms Caused by point mutations

38. Missense mutation Change of a single base pair or group of base pairs Results in the code for a different amino acid Protein will have different sequence and structure and may be non-functional or function differently

39. Nonsense mutation Change in single base pair or group of base pairs Results in premature stop codon Protein will not be able to function

40. Silent Mutation Change in one or more base pairs Does not affect functioning of a gene Mutated DNA sequence codes for same amino acid Protein is not altered

41. Frameshift mutation One or more nucleotides are inserted/deleted from a DNA sequence Reading frame of codons shifts resulting in multiple missense and/or nonsense effects Any deletion or insertion of base pairs in multiples of 3 does not cause frameshift

42. Large-scale mutations Multiple nucleotides, entire genes, whole regions of chromosomes

43. Large-scale mutations Amplification – gene duplication – Entire genes are copied to multiple regions of chromosomes

44. Large-scale mutations Large-scale deletions – Entire coding regions of DNA are removed Muscular Dystrophy

45. Large-scale mutations Chromosomal translocation – Entire genes or groups of genes are moved from one chromosome to another – Enhance, disrupt expression of gene

46. Large-scale mutations Inversion – Portion of a DNA molecule reverses its direction in the genome – No direct result but reversal could occur in the middle of a coding sequence compromising the gene

47. Large-scale mutations Trinucleotide repeat expansion – Increases number of repeats in genetic code – CAG CAG CAG CAG CAG CAG CAG CAG Huntingtons disease

48. Causes of genetic mutations Spontaneous mutations – Inaccurate DNA replication Induced mutations – Caused by environmental agent – mutagen – Directly alter DNA – entering cell nucleus – Chemicals, radiation

49. Chemical Mutagens Modify individual nucleotides – Nucleotides resemble other base pairs – Confuses replication machinery – inaccurate copying Nitrous acid Mimicking DNA nucleotides – Ethidium bromide – insert itself into DNA

50. Radiation - Low energy UV B rays Non-homologous end joining – Bonds form between adjacent nucleotides along DNA strand – Form kinks in backbone – Skin cancer

51. Radiation – high energy Ionizing radiation – x-ray, gamma rays Strip molecules of electrons Break bonds within DNA – Delete portions of chromosomes Development of tumors

52. Genomes and Gene organization Human Body – 22 autosomal chromosomes – 1 pair of each sex chromosome (XX, YY)

53. Genomes and Gene organization Components – VNTR’s– variable number tandem repeats (microsatellites) Sequences of long repeating base pairs TAGTAGTAGTAGTAG – LINEs – long interspersed nuclear elements – SINEs – short interspersed nuclear elements – Transposons – small sequences of DNA that move about the genome and insert themselves into different chromosomes – Pseudogene – code is similar to gene but is unable to code for protein

54. Viruses Not alive but can replicate themselves Contain – DNA or RNA – Capsid – protein coat – Envelope – cell membrane

55. Virus 4000 species of virus have been classified

56. HIV RNA Replication (Retrovirus) Reverse transcriptase to turn RNA into DNA Integrase incorporates into our genetic code Uses cells parts to make protein parts from mRNA genomic RNA

57. Influenza A Viral RNA replicated and transcribed for protein synthesis

58. Virus as Vectors Transduction – Using a virus vector to insert DNA into a cell or bacterium