1. Lecture 8
A toolbox for mechanistic biologists (continued)
Basics molecular genetics

2. Mass spectrometry
measures
m/z
(mass/charge)

3. Complete sequencing of a protein can be accomplished using MS/MS in conjunction with genomic information

4. What is an ideal situation for mechanistic studies of a protein of interest?
(Chapters 3 and 5)
Prokaryotic expression system
Eukaryotic expression system
Isolate that protein, generate antibodies, localize it in the cell, find counterparts, study it in vitro!

5. Density! Two different types of zonal centrifugation:
1. Velocity sedimentation
2. Equilibrium centrifugation in gradients
Density!

6. Myc- or 6His-tag affinity chromatography
6His-tagged proteins can be eluted with imidazole (His analog)
from a Ni-NTA column

7. Identify the main product on a western blot and other components (subunits) that can be co-purified

8. Restriction enzymes cleave specific DNA sequences, many of them produce ‘sticky ends”

10. If the gene is known (present in the database), then it can be amplified and cloned by PCR (Fig. 5-23)

11. The fragment can be amplified with primers that contain restriction enzyme cleavage sites, then ‘trimmed’ and inserted into a plasmid (expression vector) Fig. 5-24

12. CLONING IN BACTERIA
Fig. 5-13

13. Blue – exons (coding regions)
Green – introns (non-coding regions, which will be spliced out of the mRNA)
Unlike in yeasts, in higher eukaryotes genomic DNA does not represent continuous coding regions, thus spliced mRNA is usually taken as a starting material

14. Cloning from higher eukaryotes uses mRNA and involves a reverse transcription step (RT-PCR)
RNA → DNA transcription

15. If RT-PCR was performed with sequence-specific primers, then the trimmed fragment of interest can be inserted directly into an expression vector.
If RT-PCR was done with generic (non-specific) primers, then a cDNA library can be created and probed against known or predicted sequences.

16. Cloning into a plasmid vector

17. Cloning into lambda phage
Libraries can be screened by hybridization to a known oligonucleotide probe (see Fig. 5-16)

18. Basic Molecular Genetic Mechanisms
Bottlebrush pattern of nascent ribonucleoprotein complexes transcribed from DNA by RNA polymerase I

19. Fig. 4-1

20. The structure of DNA (4-2, 4-3) B DNA
Double stranded DNA can exist in two other forms: A and Z DNA (Fig 4-4)

21. Single- and Double-stranded DNA can be distinguished by OD260
The melting (thermal strand separation) temperature depends on the GC content

22. DNA is bendable

24. DNA has regulatory regions (promoters, enhancers) and coding regions (open reading frames)

26. Binding to the promoter is assisted by transcription factors

27. The rate of transcription at 37oC is ~ 17-20 nucleotides/s (1000 per min)
RNA polymerase is extremely processive, i.e. it remains stably associated with the DNA template all the way to the stop site.

30. Intron-Exon structure of the Fibronectin gene and alternative splicing

31. Types of RNA are involved in protein synthesis: 1. Messenger (mRNA)
2. Transfer RNA (tRNA)
3. Ribosomal RNA (rRNA)
In Eukaryotes:
RNA polymerase I - transcribes precursors 28S, 5.8S and 18S rRNAs
RNA polymerase II - transcribes all protein-coding genes
RNA polymerase III - transcribes tRNAs, 5S rRNA and small RNAs