Lecture, chapter 7 - Protein structure and function

Today we started the final chapter on the third stop in our road map, specifically on how genetic information affects protein function.

We discussed the structure of amino acids and how they are linked into polypeptides thanks to peptide bonds (a special case of covalent bonds).

We also discussed the basics of the four levels of organization in proteins, but focused specially on the secondary and tertiary levels of structure. We discussed the importance of hydrogen bonds in maintaining alpha-helices and beta-sheets, an how these are folded into 3-D structures that are crucial for protein function.

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Module 2, lab 6 (section 1) GAPC gene cloning

Click here for info on the same procedure, performed by students in section 2.

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Module 3, lab 9 (section 1) Hydrophobic Interaction Chromatography (HIC) of GFP

Click here for info on the procedure performed by students in section 2.

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Lecture, chapter 6 - Transcription

DNA looping and interaction of general and specific transcription
factors with RNA polymerase in eukaryotic cells during transcription
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Today we finished chapter 6, on transcription.

We discussed how the RNA polymerase recognizes specific DNA sequences in the promoter of prokaryotic DNA, and how it forms a transcription bubble in order to read the template strand on the DNA and synthesize the RNA molecule. We commented how the terminator causes the formation of a hairpin in the RNA transcript that causes the RNA ploymerase to "fall off" the DNA.

We highlighted the differences in transcription between prokaryotes and eukaryotes:

• the use of three kinds of RNA polymerase (I, II, and III) for transcribing different kinds of genes
• the use of general and specific transcription factors
• the role of enhancer regions in aiding the transcription process
• the need for DNA looping and the use of a mediator complex for all the transcription factors to interact with the transcription apparatus

CLICK HERE to watch a good Youtube video on transcription, or HERE for a video from the Dolan DNA Learning Center!

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Module 3, lab 9 (section 2) Hydrophobic Interaction Chromatography (HIC) of GFP

Yesterday we performed a Hydrophobic Interaction Chromatography (HIC) to separate the green fluorescent protein (GFP), produced in our bacterial cultures, from other proteins commonly found in bacteria.

A sample of bacterial culture was concentrated and then resuspended in a solution in which they were lysed. The high salt solution, containing all the proteins found in the bacteria, was then passed through a hydrophobic interaction column where molecules of GFP bound to the hydrophobic beads. The high salt solution increased the hydrophobicity of GFP by further exposing its hydrophobic amino acid residues.

A series of washes with buffers of decreasing salinity allows proteins with various levels of hydrophobicity to gradually unbind from the beads and be collected in a test tube. By switching collection tubes each time a buffer is added, different proteins can be collected. One of them was GFP and the tube in which it was collected glowed with a green color (of course).

Diagram of Hydrophobic Interaction Chromatography (HIC)

GFP molecules are represented by black triangles

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Module 2, lab 6 (section 2)GAPC gene cloning

Yesterday we started the process of cloning the GAPC gene from Arabidopsis, which we amplified via nested PCR in lab 5. The lab was divided in three main steps

• Ligation (of GAPC gene on to the pJet1.2 plasmid)
• Preparation of competent cells
• Genetic transformation of E. coli

We spent most of the lab manipulating bacteria to make them competent (i.e. get them ready to uptake extracellular naked DNA). Once this was achieved, the GAPC gene from Arabidopsis, obtained via nested PCR, was ligated to the pJet1.2 plasmid.
The plasmid was then used to genetically transform E. coli, which were spread on LB agar/Amp/IPTG plates and incubated.

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