Transcription & Translation Molecular Structure of Ion Channels.

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Transcription and Translation

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Presentation transcript:

Transcription & Translation Molecular Structure of Ion Channels

Proteins: chains of amino acids. Amino Acids: -there are only 20 different amino acids (AA). C NH 3 (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 + NH 3 H CH 2 NH 3 + Lysine: COO - C + NH 3 H CH 2 COO - Glutamic Acid: Examples:

-fold because of differences in charge along the protein. 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 into a 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 Transcription Translation Protein Summary

IntronsExons Introns: non coding regions of DNA Exons: coding regions of DNA mRNA mRNA (final) DNA Transcription “Splicing”

Why should a neuroscientist care about protein synthesis?

Ion Channel Proteins

Cell membrane is a highly durable phospholipid bilayer…….

Specialized proteins embedded within the bilayer: 1. Receptors 2. Cell Adhesion Molecules 3. 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) the the 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 are therefore 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 is a functional sodium channel? How do we know that the protein produced by the Shaker 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: ControlInhibition of gene expression