1. Genetics FROM DNA TO PROTEINS

2. How does information get from DNA to Proteins? Facts: DNA resides in the nucleus However, proteins are made in the cytoplasm In the mid 1950’s, Paul Zamecnik determines that proteins were made at the ribosome. But DNA …………………. Ribosome? How?

3. In the 1960’s it was determined that RNA was the go- between in making proteins from DNA. Spiegleman and others

4. Discovery of RNA polymerase 1960 – Hurwitz and Weiss discovered RNA polymerase RNA polymerase from E. coli is composed of five proteins (big molecule) In eukaryotes, three RNA polymerases: RNA polymerase I – makes rRNA RNA polymerase II – mRNA RNA polymerase III – small RNA molecules tRNA and one rRNA called 5S RNA

5. The Central Dogma DNA ------------->RNA------------->Protein

7. Remember all DNA is not used to make proteins. Some is used to make RNA; some functions as promoters, terminators, enhancers, etc.

8. What about proteins? Made of amino acids Each amino acid has an amino group (NH 2 ) and an acid (carboxyl) group COOH attached to a central H and a “R” group which distinguishes each amino acid. There are 20 naturally occurring aa’s.

9. R groups impart particular functions in bonding and shaping proteins. Some are polar, nonpolar, and ionized (acidic and basic). Proteins have three levels of structure: Primary – chain of amino acids lined by peptide bonds (determined by DNA) Secondary – beta sheets and alpha helices (H-bonds) Tertiary – folding by interactions between R groups Quarternary – two or more chains interacting

12. The shape of the protein determines its function.

13. What is the code? Three nucleotides code for an amino acid – the nucleotide triplet of mRNA is called a codon. Genetic code charts give the amino acids. Methionine and tryptophan have a single codon. Nearly all proteins begin with methionine. Three codons code for “stop”.

15. The redundancy of the code Since most amino acids have more than one codon, single-base changes in DNA often do not change the amino acid in the protein.

16. So how are proteins made? Transcription – DNA to mRNA Translation – Amino acids assembled to make proteins

17. Fig. 17-3a-1 TRANSCRIPTION DNA mRNA (a) Bacterial cell

18. Fig. 17-3a-2 (a) Bacterial cell TRANSCRIPTION DNA mRNA TRANSLATION Ribosome Polypeptide

19. Differences in Euk and Prok 1. In a eukaryotic cell, the nuclear envelope separates transcription from translation 2. Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA. A primary transcript is the initial RNA transcript from any gene.

20. Transcription in detail 3 steps: 1.Initiation – RNA polymerase breaks H bonds between DNA pairs and opens up the DNA to form a “transcription bubble” One strand to be copied is the antisense strand of DNA. Transcription factors help with this. 2.Elongation – RNA polymerase adds RNA nucleotides in the 5’ to 3’ direction. The elongation step has a proof-reading mechanism. e.Termination – formation of hair-pin loops of DNA. H bonds between DNA and RNA are broken and DNA rewinds.

21. Antisense strands vs Sense Strands in DNA Antisense also referred to as template strands; Sense strand also called coding strand.

23. Pre-messenger Processing mRNA is further processed in eukaryotes. 1.Splicing – remove introns, keep exons, job of spliceosomes 2.5’ cap to provide stability – 7methylguanylate cap 3.3-end – polyadenylation, a poly A tail which is a stretch of adenine bases. Required for export out of the nucleus as well as stability.

25. Job of spliceosomes

26. Translation in detail 1. Activation: The 5’ end of the mRNA transcript will bind to a ribosome on the outer membrane of the rough ER The ribosome recruits tRNAs to the site. The tRNA anticodons matches the codons on the mRNA. Aminoacyl tRNA synthetase carries out this reaction. Small subunit of ribosome attaches first and then large subunit.

27. tRNA Amino acid will attach here. Anticodon

28. tRNA synthetase ATP used for energy

29. 2.Elongation – Ribosome has three binding sites. EPA Initial binding at the P site. Add-ons at the A site. All move down as one in E site exits.

30. 3.Termination When stop codon on the mRNA sequence is reached. A release factor will bind and the complex will dissociate. Protein will fold into its conformation and enter the ER for post translational modification and packaging.

32. https://www.youtube.com/watch?v=SMtWvDbfHLo

34. Facts about insulin First protein sequenced Has an alpha chain and beta chain linked by a C-peptide Gene that codes for it is INS located on Chromosome 11 Secreted by beta cells of the pancreas Controls blood sugar

35. Modeling Insulin

36. Insulin Molecular Structure Insulin is stabilized by two covalent disulfide bonds that join the B-chain and the A-chain and a 3 rd bond in the A-chain.

37. Precursor insulin (inactive) is know as preproinsulin. The first 24 amino acids make up the ER signal sequence. As the protein is being synthesized, this signal sequence begins to emerge from the ribosome. Other proteins in the cell recognize this peptide and dock the ribosome onto the ER. As the rest of the protein is synthesized, it is directed into the lumen of the ER. From there, the preproinsulin is cleaved into four pieces as it moves through the ER to the Golgi, and to the cell surface.

38. Preparation of insulin in the cell The C-peptide is cut out of proinsulin to create the final mature insulin. As proinsulin spontaneously folds into its final 3-D shape in the ER, another protease cuts the protein as two sites: between aa 54 and 55 and between aa 89 and 90. As the C- peptide is released from the folded B-chain and A-chain complex, it floats away and is degraded.

39. Genetic Engineering of Insulin

40. 3 stages of cell signaling 1.Reception 2.Transduction 3.Response http://www.youtube.com/watch?v=qOVkedxDqQo Boseman video on cell signaling pathways Need a review of cell signaling? Campbell Chap 11

41. Fig. 11-6-1 Reception 1 EXTRACELLULAR FLUID Signaling molecule Plasma membrane CYTOPLASM 1 Receptor

42. Fig. 11-6-2 1 EXTRACELLULAR FLUID Signaling molecule Plasma membrane CYTOPLASM Transduction 2 Relay molecules in a signal transduction pathway Reception 1 Receptor

43. Fig. 11-6-3 EXTRACELLULAR FLUID Plasma membrane CYTOPLASM Receptor Signaling molecule Relay molecules in a signal transduction pathway Activation of cellular response TransductionResponse 2 3 Reception 1

44. Ligand – the signal molecule, fits like a lock and key to receptor Most ligands bind to cell surface receptors; some bind to intracellular receptors Usually induces a shape change in receptor protein’s shape

45. Types of receptors Bind with water-soluble (hydrophilic) receptors on membrane: G-Protein-linked Receptor Protein Kinase Receptor Ligand-gated Ion Channel Bind with hydrophobic receptors: Intracellular Receptors

46. Protein kinase receptors Tyrosine best understood Receptor tyrosine kinases (RTK) are enzyme membrane receptors that attach phosphates from ATP to tyrosines (Remember kinase…ATP.) Once the receptors are activated, relay proteins bind to them and become activated themselves. A receptor tyrosine kinase can trigger multiple signal transduction pathways at once

47. Fig. 11-7c Signaling molecule (ligand) Ligand-binding site  Helix Tyrosines Tyr Receptor tyrosine kinase proteins CYTOPLASM Signaling molecule Tyr Dimer Activated relay proteins Tyr P P P P P P Cellular response 1 Cellular response 2 Inactive relay proteins Activated tyrosine kinase regions Fully activated receptor tyrosine kinase 6 6 ADP ATP Tyr P P P P P P 1 2 3 4

48. Tyrosine Kinase Receptors Binding of the signal molecules causes the two polypeptides to join.

49. They are activated and act as enzymes to phosphorylate the tyrosines in the tails.

50. The receptor protein is now recognized by relay proteins, triggering different effects.

52. Ex of RTK: Insulin Signal Transduction Pathway Type 2 diabetes is accompanied by impaired insulin signal transduction. Problems here in T2 Diabetes

53. How insulin signaling works INSULIN http://faculty.plattsburgh.edu/donald.slish/InsulinReceptor4.swf http://vcell.ndsu.nodak.edu/animations/insulinsignaling/

54. How important is the Glu-4 transporters

55. The Genetic Landscape of Diabetes http://www.ncbi.nlm.nih.gov/books/NBK1667/