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59 Cards in this Set

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postulate of jacob and monod
mRNA exists
base reflects DNA
heterogeneous w/ respect to mass
associates with ribosomes
high turnover rate
cricks view of central dogma
DNA->DNA (rep)
DNA->RNA (script)
RNA->protein (lation)
RNA->DNA (RT)
other forms of RNA
participate in protein making
made by DNA-dependent RNA pol's
transcription:tightly regulated
how genes are transcribed into prokaryotes
only a single RNA pol
in e.coli the pol is 465kD
beta(prime) binds DNA
beta binds NTP and interacts with sigma
sigma recognizes promotor on DNA
a-subunits essential for enzyme activation by proteins
stages of transcription
binding RNA pol holoenzyme at promotor site
initiation of polymerization
chain elongation
chain termination
binding of pol to template DNA
pol binds nonspecific w/ low affinity, looks for promotor
sigma recognizes promotor
RNA pol holo and promotor form complex(Kd:e-6 to e-9)
pol unwinds~12pairs to open(Kd:e-14)
properties of promotors
~40bp on 5' end of transcription start site
has 2 regions:
-35region w/ TTGACA sigma binds
-10pribnow box w/ TATAAT, unwinding site
initiation of polymerization
RNA pol binds 2 places for NTP's
initation prefers to bind ATP and GTP
elongation binds 2nd NTP
3'-OH of 1st attacks a-P of 2nd to form a new phosphoester bond
when 6-10 oligo is made, sigma dissociates completing initiation
chain elongation
pol is accurate:1 error/10000bp
rate:20-50bases/sec,slower in GC rich
topo's follow pol,relieve supercoiling
supercoiling vs transcription
1.if RNA pol followed the template around the DNA, no supercoiled DNA but RNA is wrapped around helix every 10bp
2.a topo relaxing the + supercoils ahead of the advancing transcription would "relax" the DNA
chain termination
1.rho(factor prot):
ATP-dependent helicase
move along RNA,finds bubble,unwinds DNA:RNA hybrid and release RNA chain
2.specific sequences(sites in DNA)
inverted repeat,GC rich, forms loop in RNA script
6-8 As in DNA coding for Us in script
termination site for e.coli trp operon
(operon encodes for enzymes of tryptophan biosynthesis)
inverted repeats give rise to stem-loop, or hairpin structure ending in a series of U residues
rho factor mechanism of transcription termination
1.attach to recog site on mRNA
2.moves behind RNA pol
3.at RNA pol pause,unwinds hybrid
4.releases mRNA
transcription regulation in prokaryotes
1.operons:enzyme genes clustered on a chromosome
2.regulatory seq adjacent to operon determines if is "operator"
3.regulatory prot work w/ operators to control transcription of genes
induction and repression
induction:high gene synthesis due to a metabolite
repession:low gene synthesis
gratuitous inducers(IPTG):substrates that induce enzyme synthesis even tho they cant metabolize
organization of operons
linear along a chromosome
transcription control:promotor,operator(overlap)upstream from genes they control
expression:access of RNA pol to the promotor and operator.(induction activates transcription,repression is opp)
lactose and b-galactoside structures
both hexagons with 1CH2OH and 3OH but...
lactose has 2 Oxygen
beta has 1
structure of isopropyl b-thiogalactoside (IPTG)
hexagon
CH2OH
3OH
O
R:S w/ 3CH3
lac operon
lacl mutants express for lactose metabolism
structural genes controlled by - regulation
lacl pdt is lac repressor
lac repressor:DNA bindin on N-terminal and C-term binds inducer
lac operon consists of two transcription units
1.Z/Y/A controlled by Plac, that encode for b-galactosidase/b-galac permease/detoxify toxic analogs.
2.lacl w/ own Placl promotor that forms the lac repressor protein
catabolite activator protein
(+ control of lac operon)
some require accessory prot to speed transcription
catabolite activator protein(CAP) is an accessory of 22.5kD
N-term binds cAMP,C-term binds DNA
binding CAP-cAMP2 to DNA assists formation of closed promotor complex
nucleotide sequence of the lac operator
36bp of palindromic symmetry
GC bp at position 11 is symm
lac repressor protects bw -5-21+ against nuclease digestion
mechanism of catabolite repression and CAP action
glu lowers cAMP levels
cAMP is needed for CAP binding near promotors of operons whose pdts metabolize alternative energy.
binding of CAP-cAMP2 induces a severe bend in DNA about center of CAP-binding site
CAP dimer interacts w/ 2 molecules of cAMP of DNA. 2 helices of CAP goes into major groove of DNA.Binding of CAP to DNA involves H-bonding and ionic interactions bw prot and P's.
trp operon
encodes a leader sequence and 5 prot that synthesize trptophan
trp repressor controls operon
repressor binding excludes RNA pol from promotor
repressor regulates trpR & aroH operons (autogenous)
genes transcribed in eukaryotes
RNA pol I,II,II transcribe rRNA,mRNA,tRNA respectively
all have 500-700kD,2 subunits
pol II is most sensitive to a-amanitin
transcription regulation in eukaryotes
more complicated than prok
chromatin limits access of regulatory proteins to promotors
have enhancers(upstream activation sequences)
DNA looping permits multiple proteins to bind to multiple DNA sequences
TATA box in eukaryotic genes
diff # of promotors which give the percent occurence of various bases at the positions that they indicate.
metallothionein gene possesses several constitutive elements in its promotor...
as well as specific response elements MRE and GRE. BLE involve basal level expression. TRE is tumor response.
transcription factors
3 pol interact w/ promotors via transcription factors
Tf recognize and initiate transcription at promotors
some Tf (IIIA & IIIC) bind to recognition sites WITHIN the coding region
RNA pol II
(regulates mRNA synthesis)
yeast pol II:10 diff peptides
RPB1&2 are homo to e.col pol beta and beta primer
RPB1 binds DNA, 2 binds NTP
RPB1 has C-term
more RNA pol II
CTD is essential, but ISNT phosphorylated and can intiate transcription
TATA box (TATAAA) is promotor
7 Tf required
transcription initiation
TATA binding protein=TBP
binds to minor groove of DNA and pries it open creating a bend in DNA axis and unwinding w/i TATA sequence.
(all euk rely on TBP)
nucleosome structure
acK:acetylated lysine residue
meK:methylated lysine residue
PS:P-ylated serine residue
many modifiable sites and diff positions of aa
transcriptional regulation of eukaryotic genes
DNA wrapped around nucleosomes
Tf bound to enhancer or silencer
RNA pol II & GTF bound at promotor
specific Tf's stimulate and repress transcription
structural motifs
(in DNA regulatory proteins)
atomic contact bw prot residues,bases,sugar-P of DNA
contact occurs in major groove
80% reg prot:helix-turn-helix(HTH),zinc finger,leucine zipper(bZIP)
alpha helices and DNA
helices fit into major groove of B-form DNA
diameter of helix=1.2nm (same as groove)
HTH motif
2 repressors Cro and cl, and e.coli catabolite activator protein (CAP)
bind as dimers to sites on DNA
have 2 helices separated by loop w/ beta turn
C-term helix fits in major,N-term stabilizes by hydrophobic interaction with C-term helix
more HTH motif
residues 1-7 are 1st helix
residue 9 is turn maker (gly)
residues 12-20 are 2nd helix
recognition of DNA seq involves the sides of bp that face the major groove
HTH domains in sequence-specific DNA binding proteins
all are oriented the same where the view is from the rear (side opp the DNA binding face). 1st helix runs vertically down from the upper right, the turn is right center and the recognition helix runs across the center from right to left behind the domain.
Zn-finger motif
exist in almost all organisms
2 classes:C2H2 and Cx
C2H2 has Cys-x2-Cys and His-x3-His domains separated by at least 7-8 aa
Cx has 4-6 Cys residues
Zn-finger diagram
coordination of Cys and His residues to Zn is horseshoe
secondary structure is ribboned and then straight
more Zn-fingers
C2H2:folded b-strand and an a-helix
Cx:2 mini domains,4Cys to Zn followed by an a-helix.(1st helix is DNA recog, 2nd packs against the 1st)
leucine zipper motif
in almost all organisms
28-residue, w/ leu every 7th and a basic region
amphipathic a-helix and coiled-coil dimer
structure of the zipper
(and its DNA complex)
prot dimerize(homo or hetero)
basic region=DNA recog site
basic is pair of helices that wrap around major groove
homodimers recog dyad-sym DNA
heterodimers recog non-sym DNA
Fos and Jun are classic bZIP's
model for dimeric bZIp protein
2bZIP polypeptides dimerize to form a Y. Stem is the zipper and holds 2 polypeps together.Each arm is basic from one polypep, each composed of 2 a-helical segments
model for heterodimeric bZIP Tf c-Fos
c-Jun bound to a DNA oligomer with the AP-1 target sequence TGACTCA
post-transcriptional processing of mRNA in eukaryotes
slation follows scription in prok
in euk are separated(script in nuc, lation in cyto)
from nuc to cyto mRNA converts from 1mary script to mature mRNA
eukaryotic genes are split
introns bw exons
exons are 45-549 bases
introns are spliced
capping and methylation
1mary scripts(pre-m or hetero nuclear RNA) are 1st capped by guanylyl
rxn is catalyzed by guanylyl transferase
capping G residue is methylated at 7-position
adding methylation happen at 2'-O
methylation of specific sites at 5' of euk pre-mRNA is essential in mRNA maturation
cap 0: -CH3 (all euk mRNA)
cap 1:methyl added to 2'-O of 1st nucleoside after the cap (all multicellular euk)
cap 2:add a 3rd CH3 to 2'-O of 2nd nucleoside
3'-polyadenylylation
termination of scription after RNA pol has scriped past a AAUAAA seq (the polyA addition site)
10-30nucleosides past, 100-200 A resides are added to mRNA (polyA tail)
polyA polymerase adds A residues
polyA tail enhances mRNA stability
polyA addition and cleavage diagram
adds to 3' 10-35 nucleotides downstream from AAUAAA. cleavage and polyadenylylation makes a loop
consensus sequences at the splice sites in vertebrae genes
5' splice site
AG:GUAAGU
exon intron
3' splice site
Py Py..6x more-CAG:G--
intron exon
branch site and lariat
branch site:YNYRAY, Y:pyrimidine/R:purine/N:anything
lariat:closed RNA loop made by introns 5'-G to 2'-OH at A
exons join, excise lariat
lariat is unstable
splicing of pre-mRNA
(capped,polyadnylated RNA, in the form of a RNP complex is the substrate for splicing)
put exons together=mature mRNA
splicing: in nucleus
5' of intron is GU & 3' is AG
branch site is essential to splicing
importance of snRNP
(small nuclear ribonucleoprotein particles)
involved in splicing
consists of small RNA and 10 prot(some specific some not)
snRNP + pre-mRNA=spliceosome
spliceosome is size of ribos
assembly of the spliceosome
snRNP U1 & U5 bind at the 5'&3' splice sites and U2 binds at branch site
interaction bw snRNP bring 5'&3' to make lariat
transesterification rxns join the exons are catalyzed by ribozymes
events in spliceosome assembly
U1 snRNP binds at 5' then U2 at branch point. U4 is released and lariat forms freeing the 3'&5'.