UNIT VII – MOLECULAR GENETICS

1. UNIT VII – MOLECULAR GENETICS Big Campbell – Ch 17, 18, 20 Baby Campbell – Ch 10, 11, 12

2. UNIT OVERVIEW • Protein Synthesis • Regulation of Gene Expression • Prokaryotes • Eukaryotes • Mutations • Chromosomal • Gene • Cancer • DNA Technology • DNA Testing Techniques • PCR • Recombinant DNA • Extensions

3. I. PROTEIN SYNTHESIS Genotype → phenotype Central Dogma

4. I. PROTEIN SYNTHESIS, cont • History • Archibald Garrod • First to suggest genes dictate phenotype through production of enzymes • Made in 1909 after studying disease known as alkaptonuria • George Beadle & Edward Tatum • Worked with bread mold, Neurospora • Caused mutation of mold’s DNA through repeated X-ray exposure • Mutated Neurospora required enriched medium • Concluded DNA was no longer producing functional enzyme for metabolic pathways • Their work led to one gene → one enzyme hypothesis • Eventually modified to one gene → one protein • Then, one gene → one polypeptide • Now, one gene → one ??

5. I. PROTEIN SYNTHESIS, cont Working Models of Study of the Central Dogma C. elegans

6. I. PROTEIN SYNTHESIS, cont

7. II. IMPORTANCE OF RNA • Ribonucleic Acid

8. II. IMPORTANCE OF RNA, cont • Types of RNA • mRNA (_____________________) • Disposable copy of gene • “Coding RNA” • Exits nucleus via ___________ • tRNA (______________________) • Transfers amino acids to ribosome according to recipe contained in mRNA • rRNA (_______________________) • Primary component of ribosomes • Synthesized in _____________

9. II. IMPORTANCE OF RNA, cont • Non-coding RNAs …

10. III. TRANSCRIPTION Each gene contains a promoter -a specific sequence of nucleotides that marks the beginning of a gene RNA polymerase unzips the DNA and begins moving in nucleotides Nucleotides added in a _____________ direction No primer is required Only one side of the double helix is transcribed; known as the template strand Transcription continues until a termination signal is reached

11. IV. TRANSLATION mRNA is read in groups of 3 nucleotides known as a codon Sequence of three nucleotides that code for an amino acid This is also known as the reading frame Redundancy AUG Stop Codons

12. IV. TRANSLATION, cont • Transfer RNA • Function • Anticodon • Ribosomes • Function is to facilitate coupling of mRNA codon and tRNA anticodon during protein synthesis. • Made up of 2 subunits • Prokaryotic vs Eukaryotic • rRNA is transcribed from DNA, then ribosome is constructed in_______________

13. IV. TRANSLATION, cont • tRNA must bind to an amino acid • Cytoplasm of every cell stocked with all 20 amino acids required for protein synthesis • Each amino acid is joined to the correct tRNA through action of an enzyme known as aminoacyl-tRNA synthetase • There are 20 aminoacyl-tRNA synthetases • Active site fits a specific amino acid • ATP provides the energy needed to form covalent bond between tRNA & corresponding amino acid

14. IV. TRANSLATION, cont • Each tRNA anticodon must match up with the mRNA codon to insure the correct amino acid has been delivered to the ribosome. • Occurs according to base pairing rules, however there are more mRNA codons than there are tRNAs. • Certain nitrogen bases in the third position of the anticodon will base pair with more than one corresponding nitrogen base in a codon. Known as wobble.

15. IV. TRANSLATION, cont • Ribosome has 3 binding “sites” for tRNA • A Site – Holds the tRNA carrying the next amino acid to be added to the polypeptide chain • P Site – Holds the tRNA carrying the growing polypeptide chain • E “Site” – Site where tRNAs exit the ribosome • Newly added amino acids form peptide bond with carboxyl end of growing polypeptide

16. IV. TRANSLATION, cont Initiation

17. IV. TRANSLATION, cont Elongation

18. IV. TRANSLATION, cont Termination

19. IV. TRANSLATION, cont • Polyribosomes • Multiple ribosomes that translate the same mRNA multiple times • Found in both prokaryotic & eukaryotic cells

20. V. PROKARYOTIC GENE EXPRESSION • Protein Synthesis • In transcription, RNA Polymerase recognizes and binds to the promoter sequence • Transcription & translation occur virtually simultaneously

21. VI. REGULATION OF GENE EXPRESSION IN PROKARYOTES • Important adaptation for bacteria • Two basic mechanisms for metabolic control • Regulation of Enzyme Activity • Feedback Inhibition • Regulation of Gene Expression • Operons

22. VI. PRO GENE EXPRESSION REGULATION, cont • Operon Model • Operon = Promoter + Operator + all genes required for a given metabolic pathway • Operon acts as a single transcription unit • Promoter → Binding site for RNA polymerase • Operator → “On-off” switch located either close to or within the promoter • Operator controls whether or not RNA polymerase can bind to the promoter region • Therefore operator determines whether operon genes are transcribed & translated

23. VII. PRO GENE EXPRESSION REGULATION, cont • Operon Control • Operon can be turned off by a protein known as a repressor • Repressor binds to operator and prevents attachment of RNA polymerase to promoter • Repressor is a protein controlled by a gene known as a regulatory gene in a different location on chromosome; not part of operon • Expressed continuously • Always a small supply of repressor protein present

24. VII. PRO GENE EXPRESSION REGULATION, cont • Types of Operons • Inducible Operons • Operons that are usually off; that is, not usually transcribed • Can be stimulated when a specific molecule interacts with regulatory protein • Example is the lac Operon • Regulates transcription of genes required for breakdown of lactose • Typically off; bacterium is metabolizing glucose, other carbs; lactose is not present

25. VI. PRO GENE EXPRESSION REGULATION, contInducible Operons • lac Operon, cont • When lactose is available, lactose itself binds with repressor; inactivates it by changing its shape • Repressor cannot bind to regulator • Therefore, RNA polymerase is able to bind to promoter; operon is “on” • 3 enzymes required to metabolize lactose are synthesized

26. VII. PRO GENE EXPRESSION REGULATION, cont • Repressible Operons • Transcription normally occurs • Can be inhibited when a specific molecule binds allosterically to regulatory protein • Example is the trpOperon • Operon controls production of 5 enzymes required to synthesize amino acid, tryptophan when it is not available to bacterium in surrounding • Operon normally on; repressor inactive

27. VII. PRO GENE EXPRESSION REGULATION, contRepressible Operons • When tryptophan is present, it binds to the repressor of the trp operon, activating the repressor, and turning off enzyme production. • Tryptophan acts as a co-repressor, a molecule that works with a repressor protein to switch an operon off.

28. VII. PRO GENE EXPRESSION REGULATION, cont

29. VI. PRO GENE EXPRESSION REGULATION, cont • Positive Gene Regulation • In addition to repressors, some operons are also under the control of proteins known as activators • Essentially the opposite of repressors • They “turn up” an operon by making it easier for RNA polymerase to bind to DNA, therefore facilitating transcription of operon genes • In the lac operon . . . • If both glucose and lactose are available, bacterium utilizes glucose until its supplies are depleted • As glucose ↓, concentration of cyclic AMP (cAMP) ↑ • Increase in cAMP triggers release of activator protein known as CAP; CAP binds to promoter, facilitates binding of RNA polymerase to promoter of operon to enhance synthesis of enzymes of lac operon • When glucose concentration is high, decrease in cAMP results in decrease in CAP → RNA polymerase has very low affinity for lac operon promoter so lactose metabolism does not occur

30. VI. PRO GENE EXPRESSION REGULATION, cont

31. VII. EUKARYOTIC GENE EXPRESSION • Transcription • Within the promoter is a DNA sequence known as the TATA box – repeated Ts and As that identify the transcription site • Proteins known as transcription factors recognize the TATA box, bind, and allow for attachment of RNA polymerase

32. VII. EUKARYOTIC GENE EXPRESSION, cont Transcription, cont Transcription continues until polyadenylation signal (AAUAAA). mRNA is released 10-35 nucleotides downstream from polyadenylation signal although transcription continues At this point, RNA strand is known as the RNA transcript or pre-mRNA

33. VII. EUKARYOTIC GENE EXPRESSION, cont • Transcription, cont • Editing the mRNA • Each gene has long segments of non-coding DNA known as introns • Introns must be cut out of mRNA, remaining regions known as exons are spliced together, exit the nucleus, and are expressed in the translated proteins

34. VII. EUKARYOTIC GENE EXPRESSION, cont • Transcription, cont • Modifying the mRNA • 5’ end of mRNA is “capped” with a guanine nucleotide • Known as 5’ cap • 3’ end has an additional 50-250 adenine nucleotides added after polyadenylation signal • Known as poly A tail • Both modifications appear to help mRNA leave the nucleus, protect the mRNA, and facilitate the attachment of ribosomes to the 5’ end of the mRNA

35. VII. EUKARYOTIC GENE EXPRESSION, cont

36. VII. EUKARYOTIC GENE EXPRESSION, cont

37. VIII. REGULATION OF GENE EXPRESSION IN EUKARYOTES • Early in development, eukaryotic cells are totipotent • Mammalian embryos remain totipotent until 16-cell stage • Cells are described as pluripotent once extra-embryonic membranes (placenta, etc) are formed • AKA embryonic stem cells

38. As development continues, cells of multicellular organisms differentiate Differentiation due to differential gene expression in each cell, not different genes Some organisms can de-differentiate Regeneration in animals In plants, root cells can grow into mature plant IPS – Induced Pluripotent Stem Cells VIII. REGULATION OF EUK GENE EXPRESSION, cont

39. Gene expression is regulated by three mechansims Regulation of chromatin structure Regulation of initiation of transcription Post-transcriptional regulation VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont

40. VIII. EUKARYOTIC GENE EXPRESSION REGULATION, contRegulation of Chromatin Structure • 2-3 m of DNA per cell is elaborately folded • DNA wraps around proteins called histones. Charge attraction holds DNA to histones. • Cluster of histones forms nucleosome. • Stretches of DNA between nucleosomes are known as linkers

41. VIII. EUKARYOTIC GENE EXPRESSION REGULATION, contRegulation of Chromatin Structure • Folding of DNA is highly specific • Generally, the more condensed the DNA is, the less likely it is to be transcribed. • During interphase, DNA is visible as irregular clumps of chromatin. Two types: • Heterochromatin • Euchromatin

42. VIII. EUKARYOTIC GENE EXPRESSION REGULATION, contRegulation of Chromatin Structure • Modification of Histones • Acetyl (-COCH3) group added to N-end of histone “tail” • Neutralizes + charge • Histone less attracted to nucleosome, coil loosens, DNA becomes more transcribable. • DNA Methylation • Addition of methyl groups to certain bases in DNA • Most often involves cytosine • Deactivates DNA • For example, in females, inactivated X chromosome is highly-methylated

43. VIII. EUKARYOTIC GENE EXPRESSION REGULATION, contTransciptional/Translational Regulation • Regulation of Initiation of Transcription • Transcription Factors • Bind to TATA box • Form Transcription Complex that allows RNA Polymerase to bind to DNA • Enhancer Sequences • DNA sequences • May be located up to 20,000 bp “upstream” from the promoter • Bind activator proteins • Silencers • Bind repressor proteins • Work together to determine rate of transcription

44. VIII. EUKARYOTIC GENE EXPRESSION REGULATION, contTransciptional/Translational Regulation • Post-Transcriptional Regulation • Alternative RNA Splicing

45. VIII. EUKARYOTIC GENE EXPRESSION REGULATION, contTranscriptional/Translational Regulation • Post-Transcriptional Regulation, cont • Degradation of mRNA • Translation • Protein Processing & Degradation

46. VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont • Post-Transcriptional Regulation, cont • “Other” RNAs • MicroRNAs (miRNAs) • Formed from longer RNA strand that folds onto itself to create a hairpin loop • Enzyme called Dicer trims it into a short double-stranded fragment • One strand is degraded; the remaining strand can bind to any complementary mRNA • Blocks translation • Small interferring RNAs (siRNAs) • Similar in mechanism to miRNAs • Original RNA strand longer, more “hairpins”; generates many more siRNAs

47. VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont • miRNA

48. IX. MUTATIONS • Change in the nucleotide sequence • May be spontaneous mistakes that occur during replication, repair, or recombination • May be caused by mutagens; for example, x-rays, UV light, carcinogens • Two categories • Gene Mutations • Chromosomal Mutations

49. IX. MUTATIONS, cont • Gene Mutations • Point mutations – change in a gene involving a single nucleotide pair; 2 types • Substitution – Further subdivided into . . . • Silent • Nonsense • Missense • Frameshift – due to addition or deletion of nucleotide pairs Normal mRNA X

50. IX. MUTATIONS, cont • Gene Mutations & Phenotype • Traits may be described as dominant, recessive, etc . based on the effect of the abnormal allele on the organism’s phenotype • Vast majority of proteins encoded in genes are enzymes • Abnormal allele → Defective enzyme • If the enzyme produced by the normal allele is present in sufficient quantities to catalyze necessary reactions, • No noticeable effect on phenotype • Defective allele is classified as recessive • If the lack of normal enzyme production by defective allele cannot be overcome by normal allele, • Organism’s phenotype is affected • Defective allele is classified as dominant