BIOC 3400 Chapter Notes - Chapter 15-20: Poly(A)-Binding Protein, Reverse Transcriptase, Pyrosequencing
DepartmentBiochem & Molecular Biology
Course CodeBIOC 3400
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1. Bacterial 16S ribosomal
Bacterial 16S ribosomal RNA, 23S ribosomal RNA, 4S tRNA, and 5S rRNA genes are typically organized as a co-
2. blue-white screening • When using pUC18 plasmid, transformed cells are plated on agar containing antibiotic Ampicillin and X-gal
• cells containing plasmid will survive in the antibiotic
• cells containing pUC8 without DNA fragment insert will express lacZ' gene and produce β-galactosidase, the
enzyme that can splits X-gal into its component sugars, one of which is blue
• Therefore, cells containing pUC8 without insert are blue, those with inserted DNA are white
enzymatic synthesis of polynucleotide chains complementary to "template" DNA; synthesized chains are
terminated at specific nucleotide positions; resolved the fragments on a polyacrylamide gel to obtain the
4. ChIP assays Chromatin Immunoprecipitation assay;
1. DNA and associated proteins on chromatin in living cells or tissues are crosslinked (this step is omitted in
2. The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by
sonication or nuclease digestion.
3. Cross-linked DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated
from the cell debris using an appropriate protein-specific antibody.
4. The associated DNA fragments are purified and their sequence is determined. Enrichment of specific DNA
sequences represents regions on the genome that the protein of interest is associated with in vivo.
Step 1: Crosslinking: In vivo crosslinking covalently stabilizes protein-DNA complexes.
Treat cells with formaldehyde; DNA and associated proteins become cross-linked.
Step 2: Cell Lysis: lyse cells, isolate chromatin
Step 3: Chromatin Preparation (Sheering/Digestion):
sonicate or digest chromatin to 500-1000 bp fragments
Step 4: Immunoprecipitation: immunoprecipitate with antibody specific for protein of interest.
Step 5: Crosslinking Reversal and DNA Clean-up:
Reverse cross-linking, purify DNA.
Step 6: DNA Quantitation:
(quantitate the purified DNA products with quantitative PCR) use as template for PCR, or label and hybridize
6. chromatin remodeling? the dynamic modification of chromatin architecture to allow access of condensed genomic DNA to the
regulatory transcription machinery proteins, and thereby control gene expression.
7. Differences btw
transcription & DNA
-Only one DNA strand is transcribed (template strand), unidirection; newly transcribed mRNA is identical to
- RNA primer is not required.
- Only a fraction of the genome is transcribed, while in DNA replication, all of the DNA is replicated.
- Different enzyme with different properties: transcription -- RNA polymerase (slower, but high processivity);
DNA replication -- DNA polymerase ( much faster, more accurate).
- Slightly different base: U instead of T, NTPs instead of dNTPs
8. DNA footprinting
(DNase I Protection
A method of investigating the sequence specificity of DNA-binding proteins in vitro.
Detecting DNA binding site.
- Incubate protein with end labeled DNA
- Digest with DNase I
- Binding will protect a region of DNA from digestion, which can be seen as a gap on a gel image.
9. Dnase I hypersensitive
Regions of the genome particularly susceptible to experimental digestion
• Dnase I hypersensitivity is associated with actively transcribed regions
• Removal or "remodelling" of nucleosomes in these discrete regions allow the transcription apparatus access
Unit 2 -procedures
Study online at quizlet.com/_1qbecu
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10. Electrophoretic mobility shift assay,
also called Gel shift assay.
- detect binding of a protein to a sequence of DNA or RNA
- Incubate labeled nucleic acid w/ or w/o protein and separate the reactions by gel
- nucleic acid bound to protein will migrate more slowly than unbound and will appear
"shifted" in the gel
11. Factors affecting types of primers primers used for DNA sequencing depends on:
• The type of vector
• The size of the insert
12. features of eukaryotic genes Extensive post-transcriptional processing after synthesis of 'pre-RNA':
- addition of a 7-methylguanosine cap to the 5' end (serves to position the mRNA on the
- removal of introns (splicing), needs to be done very precisely!
- poly (A) tail addition by poly (A) polymerase
• Regulatory elements positioned upstream and downstream of genes
• As in prokaryotes, regulation of gene expression is at the level of transcription initiation
and control, trans-acting factors, cis-acting sites
• Transcripts are exported from nucleus before translation
13. features of prokaryotic genes • mRNAs can contain coding information for more than one protein 'polycistronic mRNAs'
• mRNA is co-linear with the DNA (no intervening sequences)
• regulation of gene expression is at the level of transcription initiation and control,
mediated by the binding of trans-acting factors at cis-acting sites
• mRNAs are used directly for protein synthesis (no post-transcriptional modification)
• Transcription and translation are coupled
14. features of pyrosequencing No labeled dNTPs, no ddNTPs, no electrophoresis!
15. Function of RNA polymerase I Transcribes pre-rRNA genes (except 5S rRNA) in the nucleolus.
16. Function of RNA polymerase II? Transcribes mRNA genes (protein-coding genes), microRNAs, and some of the small
nuclear RNA genes (snRNAs) involved in splicing
• 12 subunit polymerase complex, with many additional protein factors needed for
17. Function of RNA polymerase III? Transcribes pre-tRNA, 5S rRNA and other small RNA genes (multi-copy genes, although
typically not arranged in tandem repeats)
18. gel retardation assay, also called
Electrophoretic mobility shift assay.
This procedure can determine if a protein or mixture of proteins is capable of binding to a
given DNA or RNA sequence, and can sometimes indicate if more than one protein
molecule is involved in the binding complex.
19. How can genes be switch ON / OFF? Gene "switched on"- transcription possible
- active (open) chromatin; - unmethylated cytosines; - acetylated histones; - may bing to
other transcription factors / co-activators
Gene "switched off"- transcription impeded
- silent (condensed) chromatin; - methylated cytosines; - deacetylated histones
20. How do enhancers influence
transcription from so far away?
- Proteins bind / interact with both proximal and distal cis elements
- Transcription 'activators' bind distal regulatory sequences (e.g., enhancers)
- Multi-subunit 'co-activators' bind activators and act as bridges the activators and RNA
21. How does the actively transcribing RNA
polymerase complex pass through
arrays of nucleosomes?
• Addition of polymerase elongation factors to the transcription complex
• Some of these function as nucleosome remodelling factors, which temporarily "displace"
22. How does transcription terminate in
RNA Pol I?
Termination depends on polymerase-specific termination factors that
recognize and bind specific DNA sequences at the end of the rRNA
transcription unit. These sequences are often repetitive (e.g., mammals
have a termination sequence that is 18-bp long, repeated 10 times)
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23. How does transcription terminate
in RNA Pol III?
In vitro, RNA polymerase III is capable of terminating transcription on its
own after recognizing a stretch of T residues located shortly after the 3'
end of the mature RNA (work done with Xenopus 5S rRNA gene)
• The extent to which additional protein factors are necessary for Pol III
transcription termination in vivo is not clear.
24. How do factors influence the
-coactivator complexes; -loss of histone H1; -Histone modification ( acetylation, phosphorylation,
-Histone deacetylation, dephosphorylation, and demethylation; - corepressor complexes
25. How do transcription factors and
initiation complexes bind DNA in
the presence of nucleosomes?
• Gene-/cell-/tissue-specific protein factors that enable promoter regions to be able to accept the
"Chromatin remodeling" -Histone "tails" and histone acetylation / deacetylation or other
modification like methylation, phosphorylation, and ubiquitination
• Histone/nucleosome modification is important
26. How is 5S RNA made? multiple RNA transcripts of 5S RNA can be made before
Pol III - TFIIIA - TFIIIB - TFIIIC complex dissociates
27. How to detect labelled molecules? - Radioactively labelled molecules can be detected with X-ray sensitive film ( autoradiography) or
a radiation-sensitive phosphorescent screen (phosphorimaging)
*molecules labeled with fluorophores (dyes) with defferent emission wavelengths can be
detected with film or fluorescence detector
*molecules labeled with chemiluminescence can go through chemical reactions to generate light
signals detected with film
28. How to find the regions of DNA
bound by proteins (where RNA
polymerase and associated
29. How to find the start points of
transcription (where RNA
synthesis is initiated)?
Primer extension method
30. How to identify genomic DNA sites
bound by proteins in vivo?
Chromatin Immunoprecipitation assay.
a type of immunoprecipitation experimental technique used to investigate the interaction
between proteins and DNA in the cell. It aims to determine whether specific proteins are
associated with specific genomic regions, such as transcription factors on promoters or other
DNA binding sites
31. How to identify which proteins
are interact with DNA sequence in
gel retardation assay:
• Uses non-denaturing polyacrylamide gel electrophoresis
• Radioactively labeled DNA
• Decrease in electrophoretic mobility when DNA is bound by protein(s)
32. How to identify which sequence is
Analysis of linker scanning mutations can pinpoint the sequences with regulatory functions that
lie between the border and the transcription start site.
33. How to isolate intact
cells are embedded in low-melt agarose and digested with a protease (e.g., proteinase K) to
remove proteins, in particular, those attached to the DNA (e.g., histones)
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