Transcription & TranslationMolecular Structure of Ion Channels

Proteins: chains of amino acids.

Amino Acids:-there are only 20 different amino acids (AA).

CNH3

(amino)

COOH(acid)

Side Chain

-each has a central carbon atom.

-each has an amino group

-each has an acid group-they differ only in their side chains.

COO-

C+NH3 HCH2

CH2

CH2

CH2

NH3+

Lysine:COO-

C+ NH3 HCH2

CH2

COO-

Glutamic Acid:

Examples:

-fold because of differences in charge along the protein.

AA AA AA AA

-protein structure is determined by the sequence of AAs.

Protein Synthesis

Transcription & Translation

Begins in the nucleus………

DNA (DeoxyriboNucleic Acid):-found in the nucleus (chromosomes).

-contains the code for the synthesis of all proteins.

- Two strands of nuclei acids wrapped in a double helix.

-sequence of nucleic acids determines amino acid sequence (protein structure).

Transcription:-process of transcribing the sequence of DNA intoa sequence of RNA (ribonucleic acids).

mRNA: (Messenger RNA) resulting single strand of RNA.

Translation:-process of translating the mRNA into protein.

-occurs at ribosomes.

Nucleus

Soma

DNA

mRNA

mRNA

Transcription

Translation

Protein

Summary

Introns Exons

Introns: non coding regions of DNAExons: coding regions of DNA

mRNA

mRNA

mRNA (final)

DNA

Transcription

“Splicing”

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Why should a neuroscientist care about protein synthesis?

Ion Channel Proteins

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Cell membrane is a highly durable phospholipid bilayer…….

Specialized proteins embedded within the bilayer:

1. Receptors2. Cell Adhesion Molecules3. Ion Channels

-control the movement of ions in and out of the cell.

Studying Ion Channel Structure

Sodium Channel:

How to make a sodium ion channel:

1. Purify the protein from a rich source.

Electric eel

3. Make a single strand of DNA that matches the sequence.

(Oligonucleotide)

-attach a fluorescent probe.

2. Identify short sequences of amino acids within protein.

4. Take samples of mRNA from a source containing sodium channel mRNA.

5. Make a cDNA library from the mRNAs.cDNA:

-complementary DNA (single strand).

mRNA

Reverse Transcriptase

cDNA

-the cDNA from the sodium channel protein mRNA will contain a region that exactly matches (complements) thethe oligonucleotide.

6. Use the oligonucleotide to identify the correct cDNA.

7. From the isolated cDNA, the amino acid sequence can be determined.

Sodium Channel: 2000 amino acids long

What is the structure of the channel?

So what!

-some subunits are hydrophobic (in the membrane). -connected by hydrophilic loops (outside the membrane).

Hydrophobicity Plot:-some amino acids are hydrophobic and aretherefore found within the membrane.

The Sodium Channel

Mutant Flies

The Shaker Mutation

-mutation in potassium channel DNA.

Alternative Splicing of Shaker Locus

Shaker Splice Variants

But:

How do we know that what we have cloned isa functional sodium channel?

How do we know that the protein produced by theShaker fly is a functional potassium channel?

Get the protein to be expressed in a new organism.

Heterologous Expression

Xenopus Oocyte

What is the relationship between protein sequence and function?

Selectively mutate different coding regions.

Inject the new RNA into an organism.

Site-Directed Mutagenesis

Cell culture model:

Control Inhibition of gene expression