Term
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Definition
| controlling when and how often a given gene is transcribed |
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Term
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Definition
| Controlling the splicing and processing of RNA transcripts |
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Term
| RNA Transport and Localization Control |
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Definition
| selecting which completed mRNAs are exported from the nucleus to the cytosol and determining where in the cytosol they are localized |
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Term
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Definition
| selecting which mRNAs in the cytoplasm are translated by ribosomes |
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Term
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Definition
| selectively destabilizing certain mRNA molecules in the cytoplasm |
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Term
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Definition
| selectively activating, inactivating, degrading, or locating specific protein molecules after they have been made |
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Term
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Definition
| combinations of different proteins control a cell process |
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Term
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Definition
| turns off lysogenized phage genes |
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Term
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Definition
| turns off genes for lactose degradation |
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Term
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Definition
eukaryotes/prokaryotes
two helices at fixed angle
C-terminal helix binds to major groove of DNA
N-terminal helix contacts DNA - stabilizes/tunes
usually symmetrical dimers |
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Term
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Definition
all eukaryotes
converts body parts
resembles bacteria's H-T-H
nearly 60AA homeodomains |
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Term
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Definition
use zinc ions to stabilize
multiple types
use α-helices to recognize DNA |
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Term
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Definition
dimer of two α-helices in coiled-coil
Interact via leucines
heterodimers
allows for combinatorial control |
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Term
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Definition
homo- OR heterodimer
longer α-helix binds to major groove
can be regulated by expression of truncated H-L-H proteins
excess truncated H-L-H prevents binding to DNA |
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Term
| Gel Mobility Shift Assays |
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Definition
Protein bound to DNA will reduce its ability to move through a gel
Synthesize short, labeled fragments of DNA
mix with protein sample and run on polyacrylamide gel
Often combined with chromatography
Series of bands; each for a different DNA:protein complex
Can purify proteins from bands |
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Term
| DNA affinity chromatography |
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Definition
| Isolate DNA binding proteins |
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Term
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Definition
| method of investigating the sequence specificity of DNA-binding proteins. This technique can be used to study protein-DNA interactions both outside and within cells. |
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Term
| Chromatin immunoprecipitation |
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Definition
| 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, and possibly defining cistromes. ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers. |
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Term
| Transcriptional Repressors |
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Definition
negative control
Ex: Tryptophan Operon |
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Term
| Transcriptional Activators |
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Definition
Positive control
Many promotors are weak
Transcriptional activator
Must be bound to DNA
binds nearby DNA and RNA pol and increase transcription rate
Sometimes stabilizes transition state of RNA pol
1000x expression increase |
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Term
| + & - regulation at the same time |
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Definition
Lac operon
repressor binds in absence of lactose
CAP protein binds when glucose is low |
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Term
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Definition
Operator can be 1000s of bp away from promotor
Repressors can bind more than one operator
This leads looping of DNA
Strengthens binding of repressor
More important in eukaryotes
Effectively tethers proteins and increases probability of interacting |
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Term
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Definition
Bacteria
Subunit of RNA polymerase
Exchanged to express different set of genes
Up to 100 / genome |
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Term
| Eukaryotic Transcriptional Regulation |
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Definition
RNA pol II needs 5 transcription factors (bacteria only need sigma)
Eukaryotes lack operons
Large numbers of regulatory proteins / gene
Requires ‘mediator’ complex
Chromatin structure can regulate genes
Large ‘gene control region’ up to 50kb |
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Term
| Nucleosome Structure Hinders Expression - Dealt with by |
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Definition
Histone modification
Chromatin remodelling
Histone removal
Nucleosome replacement |
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Term
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Definition
Transcriptional regulators enhance each other
A and B each 100x =>10,000x
Result of higher order kinetics |
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Term
| Eukaryotic Repressor Proteins function by |
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Definition
competitive DNA binding
masking the activation surface
direct interaction with the general transcription factors
recruitment of chromatin remodeling complexes
recruitment of histone deacetylases
recruitment of histone methyl transferase |
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Term
| Chromatin Remodeling Complex |
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Definition
| It exhibits a DNA-dependent ATPase activity stimulated by both free and nucleosomal DNA and a capacity to perturb nucleosome structures. |
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Term
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Definition
| class of enzymes that remove acetyl groups from an ε-N-acetyl lysine amino acid on a histone. Its action is opposite to that of histone acetyltransferase. |
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Term
| Histone Methyl Transferase |
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Definition
| catalyze the transfer of one to three methyl groups from the cofactor S-Adenosyl methionine to lysine and arginine residues of histone proteins. |
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Term
| Regulatory proteins in eukaryotes |
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Definition
Not strictly activators/repressors
Can act in more than one type of regulation
Usually several proteins required
Interchangeable parts
‘committee’ of proteins |
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Term
| Eve (Drosophila Even-skipped) Protein |
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Definition
Expressed in Drosophila embryo - single cell/ many nuclei
Differential concentration across embryo
Expressed in 7 stripes
Expression controlled by concentrations of regulatory proteins
20 kb regulatory region - Modules; each responsible for one stripe
Achieved via combinatorial control - More than 20 regulatory proteins |
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Term
| Stripe 2 regulatory module |
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Definition
combionatorial control with 4 proteins
Activators: Biboid and Hunchback
Repressors: Krüppel and Giant |
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Term
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Definition
Highly regulated during different developmental stages
Gene cluster |
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Term
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Definition
erythrocytes
Through looping helps recruit
remodeling complex
Regulatory proteins
Transcription machinery
Contains barrier sequence to prevent heterochromatin sequence spread |
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Term
| Common Methods of Enzyme Activation |
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Definition
protein synthesis
ligand binding
covalent modification
addition of second subunit
unmasking
stimulation of nuclear entry
release from membrane |
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Term
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Definition
Prevent regulatory proteins from affecting other genes
Located between enhancers and promoter
Blocks crosstalk between them
Bind proteins, but mode of action not known |
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Term
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Definition
Required for tissue differentiation
DNA content of different tissues the same
=>epigenetic modifications that are inherited
Rare in prokaryotes - Exception: phase variation - Typically inherited change in cell surface protein expression |
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Term
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Definition
Sporulate to make mating cells in starvation mode
Two haploid mating types a / α
Each makes diffusible mating factor
Expression controlled by mating-type (Mat) locus |
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Term
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Definition
Model for inherited gene regulation
Model for positive feedback
Can be lytic or lysogenic
Integrase inserts lamda into bacterial chromosome
Lamda repressor / Cro - Enhance own transcription; block the other - => genetic flipflop |
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Term
| Oscillators / feedbacks / circadian clocks |
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Definition
Necessary for internal rhythm
Cell cycle
Cyclical changes in metabolic activities
Typically reset by external cues e.g. sunlight |
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Term
| DNA methylation is inherited |
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Definition
Cytosine -> 5’methyl cytosine
No effect on base pairing
In vertebrates only ‘C’
Maintenance methyltransferase acts on GC base pairs
Recoginzes methylation on other strand; adds methyl-
‘Reset’ after fertilization of egg / wave of demethlyation
Specific methyl-transferases regulate introduction of new methyl groups |
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Term
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Definition
Silencing of gene copy from one parent
If both turned on => abnormal development
E.g. Insulin-like growth factor 2 (Igf2) |
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Term
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Definition
5-methyl-C -> T deamination
=> vertegrates are GC poor / AT rich
Housekeeping genes active in germline
Promotors not methylated
Protected from deamination
=>enrichment of GC in promotors
1000-2000 bp
Ca. 20,000 in mammalian genome
Can be used to identify 5’ regions of genes |
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Term
| Inactivation of a Whole Chromosome |
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Definition
X-inactivation
Mammals : female XX ; male XY
X = 1,000 genes
Y = 100 genes
=> XX, much higher gene dose; dosage compensation |
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Term
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Definition
X-inactivation center (XIC) Encodes XIST RNA
Non-coding RNA
Binds and coats X cromosome
drives heterochromatin formation
10% of genes escape inactivation
Utilizes H2A variant
Hypoacetylation of H3 and H4; ubiquinylation of H2A; methylation H3 |
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Term
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Definition
Molecular processes are inherently random
Chaotic process; not linear
Small changes can lead to large differences in outcome
i.e. cellular processes are the result of stochastic averages
The smaller the number of molecules -> greater random effect
Small size of cells means extreme randomness
Genes flicker on and off
Life span of products and time in on/off determines signal fluctuations |
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Term
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Definition
RNA based regulatory molecules
Change conformation by binding to metabolite
Very specific, high affinity binding
Transcribed on 5’ end of a gene
Very common in bacteria
Very economical; no need to synthesize peptide |
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Term
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Definition
Splicing removes introns
Alternative splicing produces different peptides from the same RNA
Thousands of different peptides through splicing of a single gene
Very rare in bacteria
Rare in low complexity eukaryotes e.g. yeast
Common in higher eukaryotes |
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Term
| Modes of Alternative Splicing |
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Definition
optional exon
optional intron
mutually exclusive exons
internal splice site |
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Term
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Definition
Changes sequence of mRNA transcripts
Changes peptide seq and/or reading frame
Can be extensive (up to 50% of positions)
Guide RNAs - Homolgous - Bind to mRNA
Typically insertion of U
also C->U ;
A-> I - (>1000 genes in humans) - Adenosine deaminases (ADARs) |
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Term
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Definition
allows unspliced export of RNA from the nucleus
Unspliced virus RNA needed for viral particle assembly |
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Term
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Definition
Requires 3’ untranslated region - Zip code - Life span
Proteins is made where it is needed
Can establish intracellular asymetries - E.g. drosophila embryo
Regulate gene expression differently in different parts of the cell - E.e. large, polarized cells like neurons |
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Term
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Definition
| maps to a single site in the genome, corresponding to a single nucleotide pair or a very small part of a single gene |
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Term
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Definition
| inverts a segment of a chromosome |
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Term
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Definition
| deletes a segment of a chromosome |
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Term
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Definition
| breaks off a segment from one chromosome and attaches it to another |
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Term
| Restriction Endonucleases |
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Definition
Non-random
Generate fragments of specific sizes
Can be used to isolate fragment containing a gene
Isolated from bacteria - Eco RI = Escherichia coli restriction enzyme I - Bacterial DNA protected by methylation of A/C
Prevent vial infection
Tend to cut palindromes
Many (not all) make staggered cuts - ‘Sticky/cohesive’ ends can be used for ligations
Basis for recombinant DNA technology |
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Term
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Definition
forms two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor").
Requires ATP |
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Term
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Definition
| joins Okazaki fragments during DNA Replication |
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Term
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Definition
| found in non dividing cells |
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Term
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Definition
| nucleotide excision repair |
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Term
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Definition
| double-strand break repair |
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Term
| Enzyme Commission number (EC number) |
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Definition
is a numerical classification scheme for enzymes
based on the chemical reactions they catalyze.
system of enzyme nomenclature
every EC number is associated with a recommended name for the respective enzyme.
Different enzymes catalyzing the same reaction receive the same EC number. |
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Term
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Definition
Separate DNA fragments of different sizes
DNA already carries net charge; no SDS needed
Different gels for different size ranges
PAGE gels - 10-500 bp
Agarose gels - 500 -20,000 bp
Pulsed field (agarose) - 20-2,000 kb
Requires staining
Ethidium bromide
Sybr green/gold
Incorporated label |
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Term
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Definition
Double strand denatures at 100°C
Double helix can renature at <65°C - Temp / buffer control stringency
DNA:DNA (least stable) DNA:RNA RNA:RNA (most stable)
fluorescent tags
Test for presence of gene
Test for location of gene
Test for point mutation
Gene expression by blotting |
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Term
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Definition
Northern = RNA - Denaturing gel - gene expression studies - quantity - size
Southern = DNA - Denature after transfer
Western = Protein |
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Term
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Definition
Glass slides
100,000+ probes
Probes = ssDNA
Samples are labeled
Different treatments, different colors
Most useful for mRNA
Requires genome sequence
Now very cheap ! |
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Term
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Definition
Cloning = making many identical copies
Piece of DNA inserted into vector
self replicating element
Plasmid
Virus
Artificial chromosome
BAC
YAC
Library = collection of clones with different inserts |
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Term
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Definition
| Bacterial Artificial Chromosome |
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Term
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Definition
| Yeast Artificial Chromosome |
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Term
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Definition
No introns
Poly T primer for eukaryotes
Not that easy with prokaryotes |
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Term
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Definition
1st - Producing two double stranded DNA molecules
2nd - Producing four double-stranded DNA molecules
3rd - Producing eight double-stranded DNA molecules |
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Term
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Definition
Cloned mRNA/gene
Contains promotor
makes lots of mRNA
Yields protein
Most proteins at low levels in cells
Usually contain delay mechanisms (inducer)
Production is high energetic load/can be toxic
Molecular tags
His – tag (multiple histidines)
Marker protein
Signal peptide |
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Term
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Definition
Agribacterium that contains a plasmid with selectable marker
T-DNA marker; recombination which host |
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Term
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Definition
| Can dissociate in culture and regrow whole plant from single cell |
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Term
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Definition
Allows tissue/development specific off switch for a gene
Gene is replaced with functional version flanked by lox sites
when offspring is crossed with strain that contains the Cre protein (site specific recombinase), target gene is excised
Parent phenotypically normal
Offspring genetically deficient |
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Term
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Definition
Light is brought to tube by fiber optic wave guide
Light excites fluorescent die in reaction
Emitted light travels back through the wave guide to detector
Measurements are taken at the end of each cycle |
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Term
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Definition
Enzyme linked immunosorbant assay
More sensitive than just antibody tagging |
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