4. DNA makes RNA  Transcription RNA makes Proteins  Translation Information flows from genes  proteins – But not the other way! (usually)

5. Three bases in DNA code for one amino acid. The DNA code is copied to produce mRNA The order of amino acids in the polypeptide is determined by the sequence of 3-letter codes in mRNA

7. Transcription is the synthesis of mRNA from a DNA template It is similar to DNA replication in that a DNA strand is used to synthesize a strand of mRNA Only one strand of DNA is copied A single gene may be transcribed thousands of times After transcription the DNA strands rejoin

8. Get your DNA sequence – Go to NCBI: – Search this gene accession number: NM_000518.4 – Scroll and click on “Nucleotide” – Scroll down on result and copy sequence Go to this site: http://www.bioinfx.net/http://www.bioinfx.net/ Paste sequence into first box and press “submit”. What has changed?

9. Translation is the process where ribosomes synthesize proteins using the mature mRNA transcript produced during transcription. The ribosome binds to mRNA at a specific area The ribosome starts matching tRNA anticodon sequences to the mRNA codon sequence Each time a new tRNA comes into the ribosome, the amino acid that it was carrying gets added to the elongating polypeptide chain The ribosome continues until it hits a stop sequence, then it releases the polypeptide and the mRNA. The polypeptide forms into its native shape and starts acting as a functional protein in the cell.

10. If we had the DNA sequence GCAGAA The protein sequence would be: AE

11. Now lets convert your RNA sequence you made in the previous exercise to a protein. Copy your RNA sequence Go here: http://ca.expasy.org/tools/dna.htmlhttp://ca.expasy.org/tools/dna.html Paste your RNA sequence in the text box and press “Translate Sequence” Identify a few amino acids in the sequence

12. http://www.youtube.com/watch?v=983lhh20r GY&feature=related http://www.youtube.com/watch?v=983lhh20r GY&feature=related (Right click, go to hyperlink, and open hyperlink)

13. Proteins are a “necklace” of amino acids - long chain molecules The chain of molecules folds into an intricate 3D structure that is unique to each protein They provide most of the molecular machinery of cells - many of them enzymes Others play structural or mechanical roles

14. Structure is more conserved than a sequence – Similar folds often share similar functions – Remote similarities may only be detectable at structure level Interpreting Experimental Data – Locating sites of interesting mutations – Locating splice sites

15. Identify interesting sites on the protein Measure distances, angles, etc. Examine surface properties such as shape and charge Compare two protein structures

17. Hydrophobicity is a physical property in which a molecule is repelled from water During protein folding, there are hydrophobic amino acids within the protein sequence. The hydrophobic core is buried from the water which stabilizes the folded state, and the polar side chains are on the surface where they can interact with water.

18. Hemoglobin (Hb) is the iron containing oxygen transport metalloprotein in red blood cells of vertebrates Hb is made from two similar proteins that stick together. Both proteins must be present for the Hb to pick up and release O2

19. Go here: http://www.ncbi.nlm.nih.gov/Structure/CN3D /cn3d.shtml http://www.ncbi.nlm.nih.gov/Structure/CN3D /cn3d.shtml Search 1A3N Click on one of the hemoglobin molecules Identify Alpha and Beta Sheets Look at amino acid sequences What element is in the structure? How many?

22. 1.Identify the alpha and beta sheets. 2. Go to Style >> Coloring Short Cuts >> Domain. a.Why are there 4 colors? 3.Go to Style >> Coloring Short Cuts >> Residues a.What do these different colors represent? b.Go to Show >> Sequence Viewer a.Look at the 6 th residue in either B or D. b.Identify the amino acid. c.Click on the letter to determine the location in the protein. 4.Now, go to Style >> Coloring Shortcuts >> Hydrophobicity 1.Click on the 6 th residue again. 2.Is the residue hydrophobic? (Hydrophobic is brown, polar is blue) 5.Go to Style >> Coloring Short Cuts >> Element a.Which element is present, and how many? Why is there this amount?

23. A genetic disease with severe symptoms including pain and anemia (low iron) It is caused by a mutated version of the gene that helps make hemoglobin People with two copies of the sickle cell gene have the disease When the blood cells carrying the mutant hemoglobin are deprived of O2 they become sickle shaped. Carriers of the sickle cell allele are resistant to malaria - because the parasites that cause this disease are killed inside the sickle shaped blood cells.

25. Go here: http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn 3d.shtml http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn 3d.shtml Search “2HBS” - Hemoglobin S Compare both protein structures What are the differences between the proteins? Knowing what you know about the hemoglobin structure, what do you think the mutated hemoglobin may cause?

27. 1.What is the first noticeable difference in the structures when you initially view them? 2.Go to Style >> Coloring Shortcuts >> Residue 1.What is different? 3.Look at the 6 th letter in the protein sequence of B, D, F, or H. What is different? 4.Click on the letter to find the residue’s location on the protein. 5.Now, go to Style >> Coloring Shortcuts >> Hydrophobicity 1.Click on the 6 th residue again. 2.Is the residue hydrophobic? If so, how do you think this affects the structure?

28. Proteins are Amino Acid sequences. Like DNA sequences, they can be the subject of BLAST searches Protein sequences are a closer search to function and can have more results than searching a DNA sequence (remember an amino acid can represent a few different DNA sequences)

29. 1.Get the amino acid sequence from ADE2 by 1.Searching ade2 yeast mRNA and selecting the 9 th link in NCBI nucleotide search (similar to day one) 2. Copy the translation field of the CDS 2. Paste this sequence into protein blast and BLAST

30. 3.While we wait for BLAST results: Click on the ATP-grasp or AIRC super-family. Then click on one of the families contained in a colored box. A protein family describes proteins with a similar domain (which likely leads to similar function) 4. Click on the structure of the protein family to view in cn3d. 5.Take a screenshot and save the image for the flowchart figure

31. 6.Go back to the blast results and search (Ctrl+F) in the page for the fist 5 hits to “gene id”. Record the gene ID and the organism. 7.Use this to complete the table started on the first day 8. Finish the flow chart started on day two – The flow chart should indicate If/where RNA is produce If/where a protein is produce. Use the image just taken as an indicator of protein production Also make sure the PNA sequence is noted Homologs To Nucleotide sequence Gene IDOrganism Homologs to Amino Acid Sequence Gene IDOrganism PNA Binds Does Not Bind RNA? Protein? RNA? Protein?