• What is the Central Dogma?
• DNA Translation
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RNA Translation
Exercise 2 - RNA Translation
Proteins
Protein Structure Analysis
• DNA makes RNA  Transcription
• RNA makes Proteins  Translation
• Information flows from genes  proteins
– But not the other way! (usually)
• 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
• 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
• 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?
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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.
• If we had the DNA
sequence GCAGAA
• The protein sequence
would be:
• AE
• 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
• http://www.youtube.com/watch?v=983lhh20r
GY&feature=related
• (Right click, go to hyperlink, and open
hyperlink)
• 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
• 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
• Identify interesting sites on the protein
• Measure distances, angles, etc.
• Examine surface properties such as shape and
charge
• Compare two protein structures
• 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 (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
• 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?
1.
2.
Identify the alpha and beta sheets.
Go to Style >> Coloring Short Cuts >> Domain.
a.
3.
Why are there 4 colors?
Go to Style >> Coloring Short Cuts >> Residues
a.
b.
What do these different colors represent?
Go to Show >> Sequence Viewer
a.
b.
c.
4.
Now, go to Style >> Coloring Shortcuts >> Hydrophobicity
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2.
5.
Look at the 6th residue in either B or D.
Identify the amino acid.
Click on the letter to determine the location in the protein.
Click on the 6th residue again.
Is the residue hydrophobic? (Hydrophobic is brown, polar is blue)
Go to Style >> Coloring Short Cuts >> Element
a.
Which element is present, and how many? Why is there this amount?
• 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.
• Go here:
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?
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 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.
2.
Click on the 6th residue again.
Is the residue hydrophobic? If so, how do you think this affects
the structure?
• 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)
1. Get the amino acid sequence
from ADE2 by
1. 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
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.
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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
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
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The flow chart should indicate
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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 ID
Organism
PNA
Binds
Homologs to Amino Acid Sequence
Gene ID
Organism
Does Not Bind
RNA?
RNA?
Protein?
Protein?