Biochem Abnormal Amnio Gene Regulation

Front 1

do prok, euk, or both have operons?

Back 1

only prok

Front 2

LAC OPERON
name the three structural genes

Back 2

beta-galactosidase - hydrolyzes dishacch
lactose permease -brings in lactose
thiogalactoside transacetylase - acetylates disacchs that cant be cleaved by beta-galactosidase

Front 3

LAC OPERON
what are the three regulatory elements?

Back 3

operator
promoter - 2 highly conserved consensus seq's about -35 upstream
CAP(CRP)binding site

Front 4

what are the total elements of the LAC OPERON in order

Back 4

I C P O Z Y A

(I)regulatory - codes for the repressor gene

cap
promoter
operator
beta galac
permease
transacetylase

Front 5

in the LAC OPERON what does lactose act as

Back 5

the inducer that turns on the lac gene cluster that had previously been repressed

Front 6

What does the I gene do
in the LAC OPERON

Back 6

I gene makes repressor which binds the operator and shuts down transcription
NEGATIVE control

Front 7

The repressor created by the I gene has two specific binding sites
describe them

Back 7

one for lactose and the other for operator

Front 8

What happens when . .
Repressor and operator
Repressor and effector
Repressor operator and effector

Back 8

r and o = transcription blocked
E and R = repression blocked
ro and E = repression reversed

Front 9

for the LAC OPERON
describe O1 and O2

Back 9

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

Front 10

escaped synthesis

Back 10

even with repressor gene is stil transcribed a little

Front 11

Negative control of LAC OPERON includes

Back 11

repression (Igene)
and derepression(LActose to repressor)

Front 12

Positive and Catabolite control of LAC OPERON includes

Back 12

Catabolite repression(gluc represses it, reduced cAMP represses it)

Positive - increase cAMP,

Front 13

whats at -35 and -10 in E.Coli?

Back 13

-35: site of initial RNA pol attachment
-10: region of DNA melting, TATAAT

Front 14

In the absence of glucose what happens to adenylate cyclase?

Back 14

becomes phosphorylated and becomes active
makes cAMP from ATP

Front 15

in the lac operon what role does cAMP play?>

Back 15

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

Front 16

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

Back 16

- constitutive
alt operator - constitutive
alt promoter - increase expression
no adenylate cyclase - reduced expression
no cAMP binding protein - reduced expression

Front 17

what is operator constitutive

Back 17

prevent repressors from binding the operators

Front 18

Trp OPERON
What does Trp R do?

Back 18

repressor, combines with trp(co-repressor) to block operator

Front 19

Whats the difference between Trp and Lac?

Back 19

lac derepresses operon
Trp corepresses
But they are both negative control

Front 20

What is the main difference between the TRP and LAC operons?

Back 20

TRP shows attenuation (doesnt need to transcribe the full mRNA), usually stops at 162 base mRNA

Front 21

what is trp L?

Back 21

leader protein - the attenuated version of trp mRNA, contains 2 consecutive codons for trp near the 5'end

Front 22

Describe two secondary structures in pausing or attenuating the TRP operon

Back 22

two secondary structures
1) one-two hairpin pause site or three-four hairpin followed by u's(Rho-indep ish)

2)two-three

Front 23

High Trp results in termination how?

Back 23

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

Front 24

Low TRP results in no termination how?

Back 24

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

Front 25

Stringent control

Back 25

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)

Front 26

how does ppGpp get made?

Back 26

when uncharged tRNA binds A site of 50S subunit, an enz called stringent Factor is recruited
catalyses the formation of ppGpp

Front 27

how does ppGpp get made?
rxn
what does it do?

Back 27

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

Front 28

stringently controlled promoters on stable RNA have high or low affinity for RNA POL

Back 28

Low affinity

Front 29

RNA POL regulation of promoter sites

RNA POL is analogous to what in the LAC OPERON

Back 29

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

Front 30

How does ppGpp turn off ribosomes?

Back 30

blocks phosphatase which allows formation of polP which binds free ribos

without phosphorylation free ribos are degraded

Front 31

TRANSLATIONAL CONTROL
depends on

Back 31

RNA->preprotein
depends on which proteins bind shine delgarno seq
includes mRNA lifespan and
efficiency of translation

Front 32

What are R proteins or E.Coli?

Back 32

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

Front 33

what do KEY R proteins bind besides mRNA?

Back 33

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

Front 34

what will low amounts of aa's cause concerning key proteins

Back 34

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

Front 35

GAL genes
f

Back 35

genetic regulatory circuit - euk/yeast

Front 36

Function of GAL 3

Back 36

makes an inducer when galactose is present which binds gal 80 and prevents it from binding gal 4 or knocks it off gal 4

Front 37

Function of GAL 4
does what
how many domains?

Back 37

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)

Front 38

Function of GAL 80

Back 38

prevents GAL 4 from activating the genes

Front 39

Similarities between gal dnd LAC/TRP

Back 39

alpha-galactosidase like the beta in LAC
GAL2 - permease like LAC
galactose induces genes, glucose inhibits, Like LAC gluc vs lactose

Front 40

what does MEL1 do in the GAL genes

Back 40

MEL1 gene codes for aplha-galactosidase, like the b-galactosidase of lac operon, alpha cleaves melibiose to yield galactose

Front 41

What is special about GAL5

Back 41

it is constitutive(turned on all the time)

Front 42

When does catabolite repression occur in GAL ?

Back 42

when grown on gluc, even in the prescence of galac

Front 43

What are the control points found in euk not found in prok?
eg mRNA structural elements

Back 43

??euk have enhancers prok dont

Front 44

GAL 4 DNA binding site
what region does it bind?

Back 44

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)

Front 45

What method does GAL 4 use to bind DNA>

Back 45

zinc finger, 22 aa

Front 46

where does the activation domain of GAL 4 lie?

Back 46

C-terminal residues

Front 47

describe uninduced, induced and repressed in GAL

Back 47

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

Front 48

What is an analogous system to GAL gene regulation?

Back 48

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

Front 49

what other steroid receptors have zinc fingers?

Back 49

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