tuesday week 3 Flashcards
constitutive genes
ALWAYS expressed
what are genes called that are always expressed
constitutive
regulated genes
genes only expressed in the cell types and conditions in which they are required
what are genes called that are not always expressed?
regulated gene
describe in situ hybridization
single stranded labeled DNA or RNA probes detect complimentary sequences (of RNA) within tissue, cells, or chromosomes
describe/explain what a northern blot is
using RNA not DNA or proteins.
- seperate strands by mass in agar using electrophoresis
- transfer RNA strands to nylon membraine
- add probes (DNA or RNA) which will bind to RNA strands using complementary base pairing
- probes visualized using x-ray film (dots/blobs we read)
cDNA
complementary DNA. DNA synthesized using RNA as a template
whatis reverse transcriptase PCR (RT-PCR)
a method of detecting mRNA transcripts using cDNA intermediate
steps of reverse transcriptase PCR
- make cDNA using poly-T prime and reverse transcriptase
- PCR for mRNA using gene-of-interest specific primers
- gel electrophoresis to detect gene of interest
operon
several genes expressed from a common promoter with a common regulatory DNA sequence (operator)
ONLY in bacteria
operator
common regulatory sequence in operon translation
polycistronic
a single transcript containing more than one coding sequence that is translated into more than one polypeptide/protein (many rhibosomes translating on the same strand of mRNA)
ONLY in bacteria
trp
triptophan
trp operon
trp operon is expressed when tryptophan levels are low and repressed when trp levels are high (negative rienforcement loop)
low trp
the repressor is not bound by trp and does not bind to DNA. RNA polymerase transcribes the trp operon genes
high trp
trp serves as a corepressor and binds to a repressor which has a high afinity for the operator sequence in this state. tryptophan is in repressor which causes conformational change and blocks RNA polymerase from transcribing
promoters
general transcription factors + RNA polymerase bind to promoter, this initiates transcription.
promoter is always adjacent and upstream of the coding sequence
promoter activity is directional; and inverted promoter will not function
enhancers + silencers
transcription factors (activators or repressors) bind to enhancers and silences and influence the rate of transcription
how to enhancers + silencers regulate gene expression
enhancers + silencers regulate transcription through the binding of activators and repressors to their respective enhancer or silencer
enhancers + silencers can regulate at a distance, upstream/downstream, within an intron, even on another chromosome.
enhancer + silencer activity is orientation independent (can face/go any way)
mediator protein
connects (physically) enhancers + silencers to protein complex + RNA polymerase
a bridge between transcriptional activators/repressors and RNA polymerase II
how the body can modify gene expression during: DNA packing
- histone tail modifications
- histone modifying enzymes
- chromatin remodeling complex
histone tail modifications
histone tail modifications: tail modifications can alter afinity of nucleosome to the chromatin fiber or can be bound by regulatory proteins.
- modified using phosphorilation, methylation, etc
- effects tightness/loosness of DNA around nucleosome (histone octomer)
histone modifying enzymes
- histone acytl transferase (HATs) increase transcription
- histone deacetylaces (HDACs) reduce transcription
chromatin remodeling complex
ATP dependent restructing of DNA relitive to histones (complexes loosen + tighten and/or shift DNA position around nucleosomes (longer shorter ligand DNA))
ways gene expression effected by regulation during transcription
- transcription of a single gene can be regulated by many proteins - lots of things brought together + binded together to regulate a gene
- a single protein can regulate many genes (sort of like operon in bacteria) - set of different (seperate) genes share a regulatory sequence so one environmental factor (ex. increase in cortosol) will turn on all of these genes at once
epigenetic inheritance
the transmission of gene expression from parent to daughter cell(s) WITHOUT altering the DNA sequence
how does epigenetic inheritance play into gene expression/why is it important to know about?
if a TRANSCRIPTION FACTOR is part of a positive feedback loop (it activates itself), it can establish ‘memory’ of cell identity and pass that on without modifying DNA
gene expression/regulation during mRNA processing
happens specifically during splicing (usualy).
alternative splicing: different combinations of exons are spliced together to produce alternate mRNA’s that will produce different protein sequences through splicing of exon(s) or keeping of introns
alternative splicing
method of gene regulation.
different combinations of exons are spliced together to produce alternate mRNA’s that will produce different protein sequences through splicing of exon(s) or keeping of introns
how can gene expression be regulated during mRNA stability/degradation steps of transcription
sequences within 3’ UTR control the rate of mRNA degradation. the longer the AAAA sequence (tail) the longer it takes to degrade (more stable)
how can gene expression be regulated during translation
translation repressor proteins can bind to prevent translation (on mRNA post transcription ofc)
micro RNAs (miRNAs)
control mRNA stability and translation
RISC
RNA induced silencing complex
a single miRNA can inhibit translation of many mRNAs
how can gene expression be regulated during protein modification and degradation
proteasome is a protease (enzyme that degrades proteins) (26S)
ubiquitin (76aa protein) attaches to side chains of a target protein using ubiquitin ligases
what does 26s degrade
- misfolded proteins
- proteins that are regulated
- unneeded proteins
- cleaved protein fragments
ubiquitin
76aa protein that attaches to lysine side chains of a target protein using ubiquitin ligases
ubiquiting can be thought of as a way cells ‘tag’ a certain protein for recycling
poly-ubiquitinated proteins are unfolded and cleaved by the proteasome
