Exam 4: lecture 13 Flashcards
functions of signal-recognition particle
- binding to the signal peptides
2.blocking translation
3.binding to the SRP receptor on the ER membrane
how does SRP help import polypeptide into ER lumen
- SRP binds to ER signal sequence (nascent polypeptide) + blocks translation
- SRP binds to the SRP receptor; ribosome docks on membrane
- GTP binds to SRP receptor, and newly forming polypeptide moves into pore
- GTP is hydrolyzed and SRP is released
- polypeptide elongates and translocates into ER lumen
- polypeptide is completed and cleaved by signal peptidase, releasing it from the membrane
prokaryotic gene regulation: regulated genes
-controlled gene, can be turned on/off when needed
*control cell growth and cell division
*expressiom is regulated by the needed of the cells and the environment as needed (not continuously)
prokaryotic gene regulation: constitutive gene
-“on” all the time
*continuously expresse d
*housekeeping genes (those required for protein synthesis and glucose metabolism)
how does transcription regulation happen
by controlling the access of the RNA polymerase to the promoter
(controlled whether mRNA is made or not)
Transcriptional Regulation: Negative regulation
-inhibitor/repressor being made such as protein factor
-gene is ON unless a protein binds to turn it OFF
-regulatory protein turn gene OFF
-common in bacteria (can be off/on)
ex. the inhibitor blocks DNA polymerase from transcribing
Transcriptional Regulation:Positive regulation
-activator, helps the process of transcription
-gene is OFF until a protein turns it ON
-regulatory proteins turn gene ON
-occurs in eukaryotes
ex. transcription is off until an activator bind to DNA polymerase to stabilize it
what is Operon?
clusters of gene in which expression is regulated by operator-repressor protein interaction, operator region, and the promoter
contents:
promoter
repressor
operator (controlling site)
coding sequence
terminator
adjacent polycystronic closing sequences (bacteria, mtDNA) are cotranscribed to make polygenic mRNA
Operon: Inducers
chemical/ environmental agent that initates transcription of an operon
operon: induction
synthesis of gene product in repsonse to an inducer
when are inducile genes r expressed
only when induced
Lac Operon Fuction
allows cell to use lactose as a carbon and energy source break lactose into glucose and galactose
b-galactosidase: break down lactose ( the sugar binds)
transacetylase: help degrade/alter glactalose
mutation in lacO
lacO occurs upstream of lacZ and lacY and affects production of proteins downstream on the same molecule
lacO is a regulatory sequence; no diffusible product is produced
if diffusible product is produce, expect permease production in absence of lactose
mutstion in lacI
lacI produces a repressor protein
absense: repressor bind to the operator and inhibits synthesis of downstream proteins (no galactosidase/oermease are produced)
presence: repressor is inhibited by allolactose, and downstream protein synthesis occurs
mutation is promoter (Plac)
inhibit RNA polymerase bidning and protein synthesis w/wo lactose
single mutation affect all three coding gene Z,Y,A
how does positive control with catabolte activation in lac operon
-bacteria prefer glucose
glucose/no lactose low camp NO lac transcrip
no gluc/nolac high camp no lac
glucose + lactose low camp. no lac
lactose/no glucose high camp. lac tran
Operon in e coli
tryptophan
the lower level of trp, it will turned on
-based on concentration, low levels of trp, will continue expression
presence of trp, the genes are tuned off=mRNA is not transcribed and proteins r not made
absence of trp in the medium, the genes are turned on=mRNA is transcribed and proteins are made
Q. Which molecule os the co-repressor?
trp
how eukaryotic genes are regulated
time
location
dosages
eukaryotic regulations takes place at these four levels
1.pre-transcriptional: before transcription (whether it wil start
- transcriptional: controls whether transcription takes place
- translational (post-transcriptional): what happens to mRNA once it is made (translated?)
- post translational (protein regulation) whether protein cam be turned on/used
Eukaryotic regulation: 1. pre-transcriptional
-structure of DNA open/close
a. DNA methylation
cytoson—-dna methyltransferase—–> 5-methylcytosine
b.deacetylation
c. chromatin structure
- pre-transactional: DNA methylation
cytoson—-dna methyltransferase—–> 5-methylcytosine
CpG: located around promoter of housekeeping genes/ around genes frequently expressed in a cell
=CG sequence is not methylated
CG sequences in INACTIVE genes ARE methylated to suppress their expression
how are transcriptionally active chromatin regions tend to be?
hyperacetylated & hypomethylated
to do gene silencing:
histone deacetylases
ATP dependent chromatin remodolers
acetylation: add acetyle groups (red)
DNA methyltransferase: add methyl groups (yellow)
–> proteins open
deactylation: remove acetyle groups= proteins collapse
==>only methyl groups
+open/close conformation depend on availability
Eukaryotic regulation: 2.transcriptional
a. transcriptionl factors
b.repsonse/control elements
c. transactivation
d. response elements
- transcriptional: factors
activator facor: helpstablize =+ regulation (RNA transcription is increased)
repressor : destabilze protein from the promoter =-regualtion(RNA transcription is inhibited)
how is transcription regulated by a receptor?
-act as binding proteins
-once glucocorticoid enters, it oushes Hsp90, causes 2 GR dimers to bind
Eukaryotic regulation: 3. Translational
a. altenative splicing
b. mRNA stability
c.miRNA inhibition
d.alteration of translational factors
3.Translational: alternative splicing
-25k genes
shuffle exons to get different DNA
ARE=AU Rich elements: found at the 3’ UTR they control whether eukaryotic mRNA will be degraded. the more ARE’s the quicker the degradation occurs
the longer the poly a tail 3’, the stable it is
1.deadenenylation: break down tail
2.Exosome: 3’-5’ decay (chew the actual actual mRNA)
3.decapping: 5’-3’ decay
3.Translational: miRNA inhibition
-miRNA are encoded in loci other than their targets and cut to size by the ribonuclease Drosha
-they are transcribed by RNA polymerase II and their activation involves multi-step process
-expressed as longer hairpin molecules
+similar to siRNAs are generated by DICER
+associate with RISC (RNA-Inducing Silencing Complex)
+degree of complementarity to its target binding site that determines whether it will function as an miRNA/ siRNA
-22nt induce miRNA degradation or translational repression or both
What happens during SOS function?
design to stop translation when cell is in serious conditions
inactive kinase—-> active kinase—autophosphorylated
-translation is inhibited because initiator tRNAmet does not bind to the 40s subunit
Eukaryotic regulation: 4. post-translational
a. dependence on secondary effector
b. binding of protein subunits
c. chemical post translational modification
- post-translational: dependence on secondary effector
a. an example of needing a cofactor: the binding of an effector molecule such as a hormone
b. protein-protein:
the ability of the molecules that need to have a similar copy of themselves
- bind to hormone
- dimerized
ex. homodimer, heterodimer
c. posttranslational modification such as phosphorylation
-bind to phosphate to activate function
method: Northern (RNA) Blot
- take the DNA 5’TACGGA
- expose to autoradiogram
thru agarose cell
nitrocellulose
nylonmembrane
method: western (protein) blot
SDS page gel -based on weight
drawbacks:
1. takes several days
2. need protein to be isolated
3. make antibodies
method: RT-RCR(reverse transcriptase PCR)-PCR
PCR? copies a chain of DNA (vitro replication of DNA)
cycle: (30-35 times)
1. denature @ 90-95C (melt)
2.Reanneal @ 50-60C
3.Extension @ 72C replication
taq polymerase
long strands double in number w each round
exact sized increase exponentially
=2n
problem : How can you do PCR like this, when the enzymes like DNA polymerase, are killed by temperature at 94c or above
Taq polyerase (from thermus aquaticus)
what goes in the tube
primers (upstream & downstream)
dNTPS
taq polyerase
template DNA
buffer
water
PCR misprimming
When PCR primers bind to the incorrect location and allow DNA polymerase to make copies of the wrong DNA within the sample. This can be caused by too low of an annealing temperature or poorly designed PCR primers that are complementary to repetitive DNA within the genome.
RT-PCR
-uses mRNA as a template & reverse transcriptase which is an RNA polymerase
(RNA —RT—–> cDNA)—–PCR—-> lots of a specific cDNA template
allows to specifically amplify the cDNA from mRNA for cloning purposes
to determine whether that mRNA is being expressed or not
Relative expression analysis by Real TIme RT-PCR (quantitative real time)
if cDNA is detected as present, then mRNA is present to be made into cDNA and amplified
the more mRNA is present, the faster it will be amplified
DNA microarrays
used @ 100-1000 genes at once
-isolate mRNA
-convert to cDNA
-expose to light to activate fluorescent
