Exam 4: lecture 13 Flashcards by Tu Do

functions of signal-recognition particle

binding to the signal peptides
2.blocking translation
3.binding to the SRP receptor on the ER membrane

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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

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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)

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prokaryotic gene regulation: constitutive gene

-“on” all the time
*continuously expresse d
*housekeeping genes (those required for protein synthesis and glucose metabolism)

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how does transcription regulation happen

by controlling the access of the RNA polymerase to the promoter
(controlled whether mRNA is made or not)

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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

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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

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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

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Operon: Inducers

chemical/ environmental agent that initates transcription of an operon

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operon: induction

synthesis of gene product in repsonse to an inducer

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when are inducile genes r expressed

only when induced

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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

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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

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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

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mutation is promoter (Plac)

inhibit RNA polymerase bidning and protein synthesis w/wo lactose

single mutation affect all three coding gene Z,Y,A

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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

4. 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 1. bind to hormone 2. dimerized ex. homodimer, heterodimer c. posttranslational modification such as phosphorylation -bind to phosphate to activate function

method: Northern (RNA) Blot

1. take the DNA 5'TACGGA 2. 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