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control of Tx initiation:
is the most important step for determining whether genes are expressed and how much mRNA (and obviously therefore protein) is produced
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Pax6
this is a transcription factor, meaning the Pax6 gene codes for the Pax6 protein which itself is a Tx factor that controls the expression of OTHER genes that lead to the synthesis of OTHER, MORE DIFFERENT proteins! Wacky.
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transcription-control regions
specific DNA sequences that serve as binding sites for Tx factors (aka repressor and activator proteins)
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there are no introns in:
bacteria; bacteria are often organized into operons, which are genes that encode proteins all devoted to the same metabolic goal
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monocistronic mRNA
An mRNA molecule that contains the genetic information to translate only ONE protein chain (polypeptide); this is the case for most eukaryotic mRNAs
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polycistronic mRNA
mRNA that carries several open reading frames (ORFs), each of which is translated into a polypeptide; these proteins usually have a related function and their coding sequence is grouped/regulated together in an operon; bacteria
-all genes in an operon are coordinately regulated: all activated or repressed to the SAME extent
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open reading frame (ORF)
a DNA sequence that does not contain a stop codon in a given reading frame
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transcription (3 stages)
initiation, elongation, termination (PLEASE MAKE THIS A TEST QUESTION)
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PROMOTER is in the middle, RNA pol moves from left to right
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when Tx of a gene is repressed:
corresponding mRNA end encoded prtotein(s) are synthesized at a low rate
(opposite is true for when Tx of a gene is activated)
-in bacteria, repression and activation of certain genes are mostly a response to environmental changes; so the organism can change what it uses its energy for accordingly
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lac operon (positive and negative control; inducible system)
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RNA pol (talking about II in this instance)
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-catalyzes RNA synthesis
- - CANNOT FIND THE PROMOTER BY ITSELF
- -to attach to DNA strand of interest needs to associate with sigma factor
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holoenzyme
complex of RNA pol II and the sigma factor it's bound to; together, the complex can find the promoter and transcribe the gene (generally just the complete complex containing all the subunits needed for activity)
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sigma factors
- recognize the promoter SPECIFICALLY the consensus sequences at positions -35 and -10; once it is bound to transcript it recruits and attaches polymerase to itself
- -is released after pol has transcribed a few 10's of bp's
- -acts as an initiation factor required for initiation but NOT elongation
- -these factors are for bacterial translation and here are some different kinds!
- -σ70: attaches to most genes, promoter consensus:
- 10 TATAAT
- 35 TTGACA
- -σ32: attaches to heat shock genes?
- 28: motility
- 38: be stationary
- 54: NITROGEN metabolism
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translation termination
- -the main way to end transcription is to destabilize RNA pol's attachment to the DNA transcript
- at the end of a coding sequence there are often times sequences in the DNA that when translated into RNA cause the RNA strand to associate with each other (aka a stem loop is formed because the sequences are complementary)
- -after the stem loop there is often a repeat of U's which are unstable for the pairing of pol to DNA because of all the H-bonds (or A's, just in in general a repeat of U's T's or A's is more unstable than G's or C's because those are connected via 3x H-bonds)
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tryptophan synthesis is controled by negative regulation (also called a repressive system):
- -tryptophan: amino acid used for the synthesis of many proteins
- -trp operon: bacterial genome that codes for enzymes that function to make tryptophan; these enzymes participate in metabolic pathway in which end product = TRYPTOPHAN
- -so if there is enough tryptophan for the cell at a certain point, there is no need for it to keep synthesizing more of it therefore trp operon transcription/translation STOPS (is repressed)
- -repressor protein is ALWAYS expressed, meaning it's always present in the bacteria (whether it's bound to operon or not depends on other factors)
- -go to a new card
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Trp Operon if there is NO tryptophan:
- -respressor cannot bind to the operator site (aka it's inactive)
- -pol II can bind to the operon and begin Tx of the gene
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Trp Operon if there is excess tryptophan:
- -excess tryptophan binds to the respressor, converting it from its inactive to its ACTIVE form
- -it can now bind to the operator site of the operon
- -this prevents pol II from binding and subsequently Tx-ing the gene
- -tryptophan acts as a co-repressor of the trp operon
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lac operon (inducible system that uses both positive and negative control)
-encodes 3 enzymes necessary for the breakdown of lactose (sugar in milk) [so if there IS no lactose then the operon isn't expressed, because that'd be a waste of energy]
- 1) lacZ - makes B-galactosidase (enzyme)
- 2) lacY - makes permease (transporter)
- 3) lacA - makes transacetylase (aids transporter)
- -additional relevant genes: lacI makes the repressor and the operator sequence is where the repressor binds
- -is always off unless inDUCED to be turned on
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NO lactose present
- -without lactose, the repressor (lacI) is BOUND to the operator, which overlaps the Tx start site
- -no Tx of lac operon
- -etc etc
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LACTOSE and gluose present
- -if there is lactose (acts as an inducer) present, then it binds to the allosteric site of the repressor, changing its conformation and making it dissociate from the DNA
- -pol II can then bind to the promoter and Tx occurs
- -HOWEVER, if there's glucose present, the rate of Tx for the lac operon is very low (this is b/c the -10 and -35 sites where sigma70 binds differ from its ideal binding sites)
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only lactose present
- -once glucose sources have been depleted, e.coli cells respond by synthesizing cAMP
- -cAMP (small regulatory molecule) then binds to CAP protein, inducing a conformational change
- -CAP/cAMP complex can now bind to CAP site in DNA
- -the complex interacts with pol II bound at the prmoter and STIMULATES the rate of Tx
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lacI- and lacIs are mutants
- -lacI- mutants cannot produce the repressor protein (that keeps the lac operon turned off), so there is constitutive expression of said operon
- -lacI- CAN be canceled out via the insertion of a WT lacI+
- - lacIs permanently represses the expression of the lac operon; meaning the repressor is ALWAYS bound to the operator even when inducer (lactose) binds to repressor; is dominant so can't be canceled out with WT copy of lacI+
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how flexible is the DNA?
- -y-axis can be read as though it shows the level of activation
- -can use the graph to check the level of activation related to the distance between promoter and NtrC enhancer Optimal
- -can see the distance associated with the highest level of activation (or concentration of protein) is 500 bp
- -anything before and after is relatively less effective, especially after, b/c with increased distance between the two areas have a harder time finding each other
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NtrC protein (nitrogen regulatory protein C)
stimlates Tx of of this glnA gene which encodes an enzyme that makes glutamine (amino acid, - (?) charged); interacts with sigma 54 (nitrogen break down) once it's associated with the promoter
in fact, sigma 54 can't initiate the Tx UNTIL it associates with NtrC located far away
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DNA sense strand
DNA strand is a sense strand if its sequence is the same as that of a mRNA copy that is translated into protein (only difference is that T's are U's)
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DNA antisense strand
DNA on the strand opposite the sense strand (meaning corresponding mRNA is the opposite of every base in this strand)
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