Lecture 3 Outcomes
Basic structure of an amino acid and what are the different classes of amino
• Amino group, carboxyl group, R group, Hydrogen
• Different classes: nonpolar, uncharged polar, () polar (acidic), (+)
Chemistry of the peptide bond and how it is formed.
• Peptide bonds are formed by a dehydration reaction between the NH 2
(Nterminal) group of the AA and the COOH (Cterminal) group of
the other AA.
• Polypeptide: a string of amino acids
• Protein: a string of AA that has folded into the correct 3D shape
required to be functional
The four levels of protein structure.
1) Primary Structure: complete (and unique) amino acid sequence of the
protein; ex:// PheAlaLeuGlnetcetcetcetcetcetcetc 2) Secondary Structure: folding arrangement based on Hbonds between
atoms of the backbone. Most common arrangements are Alpha Helix
(a coiled, cylindrical shape) and Beta Sheets (straight side by side). A
less common arrangement is the Random Coil/Loop. *Most proteins
have a combination of all 3 arrangements simultaneously).
3) Tertiary Structure: overall 3D folding of the structure: made of 4
major interactions: ionic bonds, hydrogen bonds, hydrophobic
interactions and disulfide bridges; very flexible thus can easily change
4) Quaternary structure: arrangement when 2 or more polypeptides come
together to form a functional protein.
How are alpha helices and beta sheets formed.
Alpha: coil shaped formed when hydrogen bonds form between every NH
group of the backbone and the C—O group of the amino acid four residues
Beta: formed by the sidebyside arrangement of beta strands, made by
hydrogen bonds between atoms of each strand.
1. role of ER bound ribosomes VS. free ribosomes
• Proteins are made on ribosomes
• Ribosomes can either be free or are attached to the ER; which one depends on
the destination of the protein
• Proteins that are to be secreted or found on the plasma membrane, are
synthesized by ERassociated ribosomes; good for synthesizing mRNA to be
sent out of the cell
• All other proteins are synthesized on free cytosolic ribosomes (this is more
• Sent out=ER
• Stay in=FREE
2. points of control (POC) for regulation of protein abundance.
• Proteins are derived from PCG’s (protein coding genes)
• Lots of the genome does not code for proteins
• We are interested in protein abundance: what controls it?
• There is a difference between HOW MUCH mRNA a cell has VS. the rate at
which its being made.
• Transcription POC: Increasing the rate of transcription=increases rate of
protein abundance because there is more mRNA • Translation POC: when mRNA interacts with ribosome to make a
• mRNA decay (or turnover) POC: regulates how quickly mRNA breaks
down; some mRNA’s live for a few hours, others for a few seconds
3. factors affecting mRNA transcript abundance.
• Transcript abundance: how much mRNA is there in a cell
• Depends on the lifetime of the mRNA AND the rate of transcription
• if you have a lot of transcript, you generally have more proteins being made
• Note: transcript abundance is DIFFERENT from the rate at which
transcription is occurring
4. steps in making a Northern Blot for measuring mRNA transcript
• Transcript abundance=the specific abundance of a specific mRNA/gene
• This is called Northern Blotàwe want to measure the amount of mRNA in a
cell (southern blot=DNA)
• Step 1) we must isolate the total RNA in a cell and run it through Agarose gel
• Step 2) Then you stain the RNA so that you can see the bands
• Step 3) These bands on the black picture are rRNA because rRNA is SO
• Step 4) Now, what's the transcript abundance of the mRNA in the cell (2% of
the entire cell ie very low abundance)?
• Step 5) To manipulate the nucleic acids and probe, we transfer the total RNA
onto a thin membrane that is plastic, nylon and much more stable than the gel
• Probe is singlestranded probe, has a luminescent (radioactive) label attached
to it; we make it complementary to the RNA sequence on the membrane so
that they anneal together via hydrogen bonding in a salt solution
• Ie our probe can be specific to the opsin portion of channel rhodopsin; we
don’t need the probe to be the entire length of the mRNA, we only need at
least 1001000 base pairs
5. relative abundance of various types of RNA in typical cells.