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49 Cards in this Set

  • Front
  • Back
do prok, euk, or both have operons?
only prok
name the three structural genes
beta-galactosidase - hydrolyzes dishacch
lactose permease -brings in lactose
thiogalactoside transacetylase - acetylates disacchs that cant be cleaved by beta-galactosidase
what are the three regulatory elements?
promoter - 2 highly conserved consensus seq's about -35 upstream
CAP(CRP)binding site
what are the total elements of the LAC OPERON in order

(I)regulatory - codes for the repressor gene

beta galac
in the LAC OPERON what does lactose act as
the inducer that turns on the lac gene cluster that had previously been repressed
What does the I gene do
I gene makes repressor which binds the operator and shuts down transcription
NEGATIVE control
The repressor created by the I gene has two specific binding sites
describe them
one for lactose and the other for operator
What happens when . .
Repressor and operator
Repressor and effector
Repressor operator and effector
r and o = transcription blocked
E and R = repression blocked
ro and E = repression reversed
for the LAC OPERON
describe O1 and O2
O1 - seq of the operator just downstream of promoter, ~24 nt's,
O2 - within Z
Repressor binds O1 and O2 looping DNA, RNA pol can bind(affinity actually increases with loop) simultaneously but will not work
escaped synthesis
even with repressor gene is stil transcribed a little
Negative control of LAC OPERON includes
repression (Igene)
and derepression(LActose to repressor)
Positive and Catabolite control of LAC OPERON includes
Catabolite repression(gluc represses it, reduced cAMP represses it)

Positive - increase cAMP,
whats at -35 and -10 in E.Coli?
-35: site of initial RNA pol attachment
-10: region of DNA melting, TATAAT
In the absence of glucose what happens to adenylate cyclase?
becomes phosphorylated and becomes active
makes cAMP from ATP
in the lac operon what role does cAMP play?>
builds up in the absence of gluc due to adenylate cyclase and binds CAP (catabolite activator protein) an allosteric regulator. this new CRP formed binds promoter and enhances RNA POL binding
For the LAC operon what is the outcome of the following defects

no i protein
altered operator
altered promoter
no adenylate cyclase
no cAMP binding protein
- constitutive
alt operator - constitutive
alt promoter - increase expression
no adenylate cyclase - reduced expression
no cAMP binding protein - reduced expression
what is operator constitutive
prevent repressors from binding the operators
What does Trp R do?
repressor, combines with trp(co-repressor) to block operator
Whats the difference between Trp and Lac?
lac derepresses operon
Trp corepresses
But they are both negative control
What is the main difference between the TRP and LAC operons?
TRP shows attenuation (doesnt need to transcribe the full mRNA), usually stops at 162 base mRNA
what is trp L?
leader protein - the attenuated version of trp mRNA, contains 2 consecutive codons for trp near the 5'end
Describe two secondary structures in pausing or attenuating the TRP operon
two secondary structures
1) one-two hairpin pause site or three-four hairpin followed by u's(Rho-indep ish)

High Trp results in termination how?
RNA POL transcribes regions three and four while ribosome translates one and two,
Ribosome covers region 2 so it can't pair with region three, thus the three-4 hairpin is formed which is a rho indep termination
Low TRP results in no termination how?
ribosome stalls on one because it is waiting for a tRNA~trp which is in low concentrations
stall allows RNA POL to be released from the pause one-2 hairpin and transcribe region 3,
2-3 is more stable than 3-4 so 3-4 wont form
Stringent control
inhibiting genes assoc with ribosome biosynthesis upon aa starvation
however, rate of total mRNA syn is only slightly reduced and some mRNA's are actually stimulated (like the TRP operon)
how does ppGpp get made?
when uncharged tRNA binds A site of 50S subunit, an enz called stringent Factor is recruited
catalyses the formation of ppGpp
how does ppGpp get made?
what does it do?
GDP + ATP + uncharged tRNA in A site of ribo --> ppGpp + AMP

ppGpp either binds RNA pol and or acts as second messenger
Turns off rRNA
stringently controlled promoters on stable RNA have high or low affinity for RNA POL
Low affinity
RNA POL regulation of promoter sites

RNA POL is analogous to what in the LAC OPERON
region 40-98 bp upstream of start has a pseudo promoter which RNA POL binds, another RNApol binds the first and can then reach over and positively activates transcription of an RNA pol downstream.

RNA POL can act like CAP in the LAC OPERON
How does ppGpp turn off ribosomes?
blocks phosphatase which allows formation of polP which binds free ribos

without phosphorylation free ribos are degraded
depends on
depends on which proteins bind shine delgarno seq
includes mRNA lifespan and
efficiency of translation
What are R proteins or E.Coli?
ribosomal proteins, when they exceed rate at which rRNA is made they begin to accumulate,
Certain KEY ones then bind SD seq's of mRNA's that make R-proteins(almost like feedback inhibition)
this ensures ribosomal proteins are not made faster then they can be used in making ribosomes
what do KEY R proteins bind besides mRNA?
rRNA, competition between SD and rRNA binding,
so when not in excess, key proteins will participate in the formation of ribosomes in preference to binding R-Protein mRNA
what will low amounts of aa's cause concerning key proteins
will cause key protein inhibition to turn on,

regulation of ribosome production reflects regulation of rRNA syn
rRNA syn is regulated by stringent control

key prot inhibition is off when aa's are normal
GAL genes
genetic regulatory circuit - euk/yeast
Function of GAL 3
makes an inducer when galactose is present which binds gal 80 and prevents it from binding gal 4 or knocks it off gal 4
Function of GAL 4
does what
how many domains?
constitutive, binds upstream UAS and activates the 5 GAL gene's transcription

has two functional domains
DNA bindign and transcription activation region (recall TF's)
Function of GAL 80
prevents GAL 4 from activating the genes
Similarities between gal dnd LAC/TRP
alpha-galactosidase like the beta in LAC
GAL2 - permease like LAC
galactose induces genes, glucose inhibits, Like LAC gluc vs lactose
what does MEL1 do in the GAL genes
MEL1 gene codes for aplha-galactosidase, like the b-galactosidase of lac operon, alpha cleaves melibiose to yield galactose
What is special about GAL5
it is constitutive(turned on all the time)
When does catabolite repression occur in GAL ?
when grown on gluc, even in the prescence of galac
What are the control points found in euk not found in prok?
eg mRNA structural elements
??euk have enhancers prok dont
GAL 4 DNA binding site
what region does it bind?
binds UAS(upstream activating seq)similar to enhancers in higher euk because they can function in any orientation (3' or 5' to the gene)(ie can be either strand)
What method does GAL 4 use to bind DNA>
zinc finger, 22 aa
where does the activation domain of GAL 4 lie?
C-terminal residues
describe uninduced, induced and repressed in GAL
uninduced - GAL4 blocked by GAL 80

induced GAL 4 binds TBP at TATA box

REpressed - catabolite repressor protein binds GAL4 which is bound by gal 80
What is an analogous system to GAL gene regulation?
steroid hormone regulation

steroids bind cytosolic receptors which enter nucleus and activate transcription,

the glucocorticoid Hormone receptor binds to GRE which is exactly analogous to UASgal
what other steroid receptors have zinc fingers?
testosterone and progesterone, bind SRE's (steroid response element) similar to GRE's, but they behave like true enhancers and can be anywhere in DNA