Post-transcriptional regulation
What does polysome profiling tell you about gene expression at the translational level?
How many ribosomes are associated with each transcript (translation efficiency)
What two physical properties are used in 2D gel electrophoresis to separate proteins?
Isoelectric point and molecular weight
Which of the following is NOT a high-throughput technique used in proteomics? Tandem mass spectrometry, quantum dot-tagged microbeads, 2D gel electrophoresis, reverse phase protein arrays
2D gel electrophoresis
What is the difference between forward and reverse genetics?
Forward genetics starts with a phenotype of interest and attempts to find the casual gene; reverse genetics starts with a locus of interest and attempts to find the resultant phenotype
Explain X-ray crystallography.
Technique used to determine atomic resolution structure of crystallized molecules such as proteins.
Name two examples of post-translational modifications.
Possible answers: Proteolytic cleavage, chemical group addition (methylation, acetylation, phosphorylation, etc)
Name two types of protein attachment strategies.
Possible answers: Diffusion, adsorption, covalent attachment, affinity
Name 2 reasons why protein microarrays are more difficult to design than nucleic acid microarrays?
Nucleic acid microarrays are based on complementary base pairing and hybridization with nucleic acid probes, which is predictable. An antibody protein chip, for example, will require the production of specific antibodies for each protein. Proteins also have more complex 3D structures.
What are the minimum requirements in a functional CRISPR-Cas9 vector to enable genome editing?
Cas9 coding sequence, gRNA sequence (must be complementary to target DNA with PAM sequence), origin of replication. Optionally a selectable marker.
Which of the following is NOT a technique that can be used for protein sequencing? What is it used for instead? Nanopore, Quantum-Si Platinum Pro, Protein antibody arrays, Encodia
Protein antibody arrays - Used for detecting protein abundance or activity, does not provide amino acid sequence
Describe 2 advantages of regulating gene expression at the translational level rather than the transcriptional level.
Possible answers:
Rapid response - no need to wait for new mRNA, can immediately begin or halt protein production
Spatially tunable - Transcription is localized in the nucleus. Translational regulation can occur anywhere in the cell
Name one pro and one con of using classical 2-D gels in proteomics.
Pro - Can separate up to 2000 proteins with post-translational modification detection
Con - Poor performance for very acidic/basic proteins, membrane proteins, or low-abundance proteins
Name and explain one method of reading the results of a protein-chip experiment.
Possible answers:
Fluorescence (probe the chip with fluorescently tagged protein or other molecule)
SELDI (use a laser to ionize proteins and then run through mass spec)
Surface plasmon resonance (detect microscopic changes on surface of chip that are the result of captured proteins)
Give one example of a dominant mutation and one example of a recessive mutation. Explain how they affect gene function.
Possible answers:
Dominant - hypermorph (gain of function), neomorph (new function), antimorph (negative interaction with WT)
Recessive - hypomorph (reduced/loss of function), haploinsufficient (weaker heterozygous phenotype)
Name and describe 3 different applications of mass spectrometry discussed in this class.
Possible answers (there may have been more i forgot)
Tandem affinity purification (TAP) followed by mass spec for protein-protein interaction
MALDI-TOF for determining SNP identity at specific position
MALDI (TOF or ion trap) for protein identification
ICAT for proteomic differential expression
Tandem MS/MS for sequencing peptides
SELDI for reading results of protein chip
List EVERY step of regulation that occurs after transcription of an mRNA.
Capping, polyadenylation, splicing, RNA editing, nuclear export, subcellular localization, translation initiation and repression, mRNA stability and degradation, RNAi
Compare the data yielded by ribosome profiling (Ribo-seq) versus that yielded by polysome profiling.
Ribo-seq uses ribosome footprinting to identify the exact location and identity of translating transcripts, revealing uORFs and pausing. Polysome profiling gives a global view of how many ribosomes are translating a given transcript but lacks resolution of specific binding sites.
Describe 2 advantages of using classical gel-based protein arrays over others such as glass.
Possible answers:
Higher protein capacity due to 3D matrix
Homogenous water environment that prevents aggregation and denaturation and facilitates protein interactions
Name the three methods of mutagenesis discussed in class and one example of each.
Chemical mutagenesis - EMS, ENU
Radiation mutagenesis - X-rays, lambda-rays, fast neurons
Insertional mutagenesis - transposon, T-DNA
Explain optogenetics and what it is used for.
Use of light-sensitive proteins to control gene expression. Uses light-controlled TFs and a split activator.
How can reporter genes be used to evaluate post-transcriptional regulation?
Can isolate and test elements like those affecting localization, stability, or translation. For example, can check different splicing patterns by inserting different introns and assessing how this affects reporter expression.
Name 2 pros and 2 cons of Y2H matrices.
Pros: all baits and preys tested simultaneously, no sequencing required
Cons: full length ORFs mask interacting domains, may be toxic, and/or have problems with transport to nucleus. Also, can have false positives
Explain quantum dot-tagged microbeads, what they are used for, and 2 advantages of this technique.
Polymer microbeads conjugated to a quantum dot barcode. They can be mixed in a single assay to allow multiplexed detection of different proteins. They allow for massively parallel analysis, have the flexibility to interrogate thousands of proteins without needing a new chip, and are used in solution, which is closer to a protein’s native environment.
Explain TALENs and how it differs from other techniques like CRISPR and ZFNs.
Transcription activator-like effector nucleases, used for specific genome editing.
2 TALEN proteins are designed to bind opposite strands of a target DNA sequence with a spacer. When both bind, the FokI domains dimerize and induce a DSB, which is repaired with NHEJ or HDR.
Easier to design than ZFNs, but more laborious to construct and don’t require a PAM sequence.
You are investigating a newly discovered RNA-binding protein (RBP-X) that appears to regulate translation of stress-response mRNAs under cold conditions in cotton.
Your goals are to:
Confirm whether RBP-X binds specific mRNA targets.
Determine how RBP-X affects translation of those targets under stress.
Characterize the structure-function relationship of RBP-X.
Identify whether RBP-X activity is reversible or temporally regulated.
Design an experimental approach that incorporates at least three different techniques discussed in this course to address these objectives.
idk lets discuss