Lecture 11 Flashcards
What method can be used for promoter analysis? What does it do?
Gibbs Sampler (motif identification)
Searches for statistically most probably motifs in unaligned sequences
What methods are used for determining transcription factor binding site in silico?
- Word counting methods
- Gibbs sampling
- Phylogenetic footprinting/ comparative genomics
Describe the steps of Gibbs Sampling
- Set width for motif
- Choose random position for the start of the motif in all but one sequences
- Estimate the amino acid (or nt) frequencies in the motif columns of all but left-out sequence
- Estimate background frequencies (nt0): frequencies of nt (or aa) in positions that are not considered the motif
- Scan out the left out sequence and estimate probability of finding the motif at any position
- Calculate odds score ratio for each position (a= pobserved / p background) - Add up all above odds score and divide the odds score for each position by the total to obtain probability that motif is at that position
- These probabilities used as weights to decide a probable location of the motif in the left out sequence
Repeat > 100 times
What is the goal of Gibbs sampling?
Find the most probable patterns common to all of the sequences by sliding them back and forth until the ratio of the motif probability to the background probability is a maximum
How was Gibbs Sampler modified?
- search multiple motifs
- seek pattern in only fraction of input sequences (bc not all genes regulated by same TF or regulatory mechanism)
- Look motifs of different widths
- Avoid suboptimal solution by shifting current alignments a certain number of positions to right and left
For what is the hypergeometric p-value used?
To ask if there are GO categories enriched in my cluster
Name 4 methods to study protein-protein interactions
- Classic biochemical (chromatographic) methods
- yeast two hybrid followed by clone sequencing
- Affinity purification (TAP tagging then mass spectrometry)
- interologs or BioID
How does Y2H work?
Attach Gal4 binding domain to bait protein
Separate Gal4 activation domain and attach to prey protein
if bait and prey interact in vivo in nucleus of yeast, Gal4 fxn is reconsittuted and drive expression of reporter gene:blue yeast colonies
Problems of Y2H:
-Assay done in yeast, so might not get modifications necessary for protein funtion
-overexpressing protein and targetting to nucleus
-not well for membrane proteins
Is Y2H useful for membrane proteins?
no
Is Y2H useful for transient interactions?
Yes for transient binary interactions
What is TAP?
Tandem affinity purification
How does TAP tagging work?
immunoprecipitation-based purification technique for studying protein–protein interactions. The goal is to extract from a cell only the protein of interest, in complex with any other proteins it interacted with. TAP uses two types of agarose beads that bind to the protein of interest and that can be separated from the cell lysate by centrifugation, without disturbing, denaturing or contaminating the involved complexes. To enable the protein of interest to bind to the beads, it is tagged with a designed piece, the TAP tag.
The original TAP method involves the fusion of the TAP tag to the C-terminus of the protein under study. The TAP tag consists of three components: a calmodulin binding peptide (CBP), TEV protease cleavage site, and two Protein A domains, which bind tightly to IgG (making a TAP tag a type of epitope tag).
What are other types of arrays used for protein study?
- Protein Microarrays
- Glycan Arrays (arrayed 100s carbohydrates onto slides as a tool to understand carbohydrate biology)
Why study glycosylation?
-regulatory mechanisms
-carbs key structural support in plant biology
