Study Guide MBMB 451A Fall 2007
Part Two
Transcription and its Regulation
1. Explain the differences between activated and basal transcription both in
terms of DNA sequence elements and protein factors.
2. Describe the properties of the general transcription factors of RNA
polymerase II, I and III. What is TBP and what general transcription factors
have TBP as a component? What enzymatic activities do some of the
transcription factors possess and how do they facilitate transcription? Describe
TAFs and if they are involved in activated or basal transcription.
3. What are three models of the molecular events that lead to activation of
transcription?
4. Structural organization of promoter-specific transcription: domain structure
and function.
5. You will need to know about the different types of protein structural
domains that are used to recognize specific DNA sequences (Zn finger, bHLH,
and so). What are their distinctive properties, in what transcription factors are
they found, and do they have any required cofactors (metals, ligands, etc)?
6. What is the promoter structure for genes transcribed by mRNA, tRNA,
rRNA, 5S rRNA, and U6 snRNA?
7. What are transcriptional coactivators?
8.What are the different ways to regulate the activity of transcription including
merely regulating the level of the transcription factor present in the cell?
9. Describe how steroid receptors function and their structure.
10. What are the two kinds of transcription elongation factors? How do some
of these factors enhance transcription elongation?
11. What are three classes of activation domains?
Protein Methods
1. Describe the different methods used to lyse cells and to make a soluble
protein extract. How is precipitation used to help purify a protein and what
substances are used for this purpose?
2. What are the principles of ion-exchange chromatography and how does it
work in protein purification? What is a linear versus a step gradient? What is
the difference between a weak anion exchange resin and a strong anion
exchange resin? What kind of materials are used to make the solid support for
column chromatography (cross-linked agarose, …).
3. Describe the different types of affinity chromatography and how they work.
4. How does gel filtration or exclusion chromatography work? What is the
exclusion limit or volume? How do you attach something to your resin of your
choice?
5. What is HIC chromatography?
6. Explain discontinuous gel electrophoresis and how it is used to separate
proteins by size. What is an SDS- micelle and what is its role in gel
electrophoresis?
DNA Recombination
1. Explain how DNA recombination can occur with DNA starting with a nick
or with their being a double stranded break in DNA? (see lecture notes) How
does one get reciprocal versus patch recombination in this manner?
2. What is the Holliday junction? What is a D-loop?
3. What is a synaptonemal complex, and at which stage in meiosis does it
occur. Describe the structural features of this complex and its known protein
components.
4. Explain how the RecBCD complex works and the role of the chi sequence?
At what stage does RecA come into the process? What are the functional and
structural properties of the RecA protein?
5. How do the RuvA, B, and C proteins function to resolve the Holliday
junction? What are their structural properties?
6. How does specialized recombination work? Explain how recombinases
work and their intermediates that are formed in the process.
7. What is Cre-lox?
RNA processing
1. Describe the different features of the cap structure of mRNA and the
associated proteins responsible for making these modifications. What does the
5’ cap for the mRNA and in what way does it function (stabilization,
translation)?
2. Be able to describe the process of mRNA splicing, the components of the
spliceosome and how they are involved in splicing. What is a
transesterification reaction and how does it relate to splicing? What are an
exon and an intron, and the conserved features of the 5’ and 3’ splice junctions?
In what organisms are introns found and are there general lengths for exons and
introns? What is the connection between exons and the modular structure of
proteins?
3. Describe the process of self splicing and the differences between group I
and II introns. Make sure to discuss any nucleotide requirement. What is a
ribozyme?
4. What is alternative splicing? What are other ways that you can get more
than one protein product from essentially the same region of DNA? What is
trans-splicing and RNA editing?
5. Describe the process of making the mature 3’-end of a mRNA. Make sure
to know any sequence requirements in the mRNA and the proteins involved in
this process. Are there generally transcriptional stop sites in eukaryotic mRNA
genes?
6. What does siRNA or RNAi stand for? Understand how siRNA f unctions,
particular in regards to dicer activity and targeting by the RITS complex.
7. Describe how rRNA and tRNA are processed, highlighting cleavage events,
methylation, nucleotide additions, and other kinds of modifications.