VISUALIZING THE CENTRAL DOGMA

Day 3 - SAMS ProgramWill Foran

Sam Sanford

Objectives

• What is the Central Dogma?• DNA Translation• Exercise 1 - DNA Transcription• RNA Translation• Exercise 2 - RNA Translation • Proteins• Protein Structure Analysis• Exercise 3 – 3D Structure of 1A3N • Exercise 4 – 1A3N and 2HBS Comparison

What is the Central Dogma?

The Central Dogma

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

proteintranscription translation

mRNADNA

replication

DNA Contains Codes• 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

RNA vs. DNA

DNA Transcription• 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

Mini Exercise: DNA Transcription

• 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/• Paste sequence into first box and press “submit”.• What has changed?

RNA Translation

• 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.

Amino Acid Codes• If we had the DNA

sequence GCAGAA• The protein sequence

would be:• AE

RNA Translation Exercise

• 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.html• Paste your RNA sequence in the text box and

press “Translate Sequence”• Identify a few amino acids in the sequence

DNA Transcription and Protein Assembly Video

• http://www.youtube.com/watch?v=983lhh20rGY&feature=related

• (Right click, go to hyperlink, and open hyperlink)

Proteins

• 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

Why Examine Protein Structure?

• 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

Structure Analysis

• Identify interesting sites on the protein• Measure distances, angles, etc. • Examine surface properties such as shape and

charge• Compare two protein structures

Protein Structure

Hydrophobicity• 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.

Hemoglobin Background Information

• 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

Exercise 3 - Protein Structure

• Go here: 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?

Search for “1A3N”

Select Display as “Single 3D Structure” and press “View Structure”

Exercise 3 Questions1. 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 6th 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 >> Hydrophobicity1. Click on the 6th residue again. 2. Is the residue hydrophobic? (Hydrophobic is brown, polar is blue)

5. Go to Style >> Coloring Short Cuts >> Elementa. Which element is present, and how many? Why is there this amount?

Sickle Cell Anemia • 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.

DNA Level of Sickle Cell Anemia

Exercise 4 - Protein Structure Comparison

• Go here: http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.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?

Select “Asymmetric Unit” and then “View Structure”

Exercise 4 Questions1. 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 6th 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 6th residue again. 2. Is the residue hydrophobic? If so, how do you think this affects the

structure?

Blasting Proteins

• 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)

Blasting ADE2 AA Seq1. Get the amino acid sequence

from ADE2 by1. Searching ade2 yeast mRNA

and selecting the 9th 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

Blasting ADE2 AA Seq3. 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

Figures6. 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 sequenceGene ID Organism

Homologs to Amino Acid SequenceGene ID Organism

PNA Binds Does Not Bind

RNA?

Protein?

RNA?

Protein?