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59 Cards in this Set
- Front
- Back
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postulate of jacob and monod
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mRNA exists
base reflects DNA heterogeneous w/ respect to mass associates with ribosomes high turnover rate |
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cricks view of central dogma
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DNA->DNA (rep)
DNA->RNA (script) RNA->protein (lation) RNA->DNA (RT) |
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other forms of RNA
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participate in protein making
made by DNA-dependent RNA pol's transcription:tightly regulated |
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how genes are transcribed into prokaryotes
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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 |
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stages of transcription
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binding RNA pol holoenzyme at promotor site
initiation of polymerization chain elongation chain termination |
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binding of pol to template DNA
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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) |
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properties of promotors
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~40bp on 5' end of transcription start site
has 2 regions: -35region w/ TTGACA sigma binds -10pribnow box w/ TATAAT, unwinding site |
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initiation of polymerization
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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 |
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chain elongation
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pol is accurate:1 error/10000bp
rate:20-50bases/sec,slower in GC rich topo's follow pol,relieve supercoiling |
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supercoiling vs transcription
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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 |
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chain termination
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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 |
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termination site for e.coli trp operon
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(operon encodes for enzymes of tryptophan biosynthesis)
inverted repeats give rise to stem-loop, or hairpin structure ending in a series of U residues |
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rho factor mechanism of transcription termination
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1.attach to recog site on mRNA
2.moves behind RNA pol 3.at RNA pol pause,unwinds hybrid 4.releases mRNA |
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transcription regulation in prokaryotes
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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 |
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induction and repression
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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 |
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organization of operons
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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) |
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lactose and b-galactoside structures
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both hexagons with 1CH2OH and 3OH but...
lactose has 2 Oxygen beta has 1 |
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structure of isopropyl b-thiogalactoside (IPTG)
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hexagon
CH2OH 3OH O R:S w/ 3CH3 |
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lac operon
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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 |
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lac operon consists of two transcription units
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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 |
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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 |
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nucleotide sequence of the lac operator
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36bp of palindromic symmetry
GC bp at position 11 is symm lac repressor protects bw -5-21+ against nuclease digestion |
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mechanism of catabolite repression and CAP action
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glu lowers cAMP levels
cAMP is needed for CAP binding near promotors of operons whose pdts metabolize alternative energy. |
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binding of CAP-cAMP2 induces a severe bend in DNA about center of CAP-binding site
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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.
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trp operon
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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) |
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genes transcribed in eukaryotes
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RNA pol I,II,II transcribe rRNA,mRNA,tRNA respectively
all have 500-700kD,2 subunits pol II is most sensitive to a-amanitin |
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transcription regulation in eukaryotes
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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 |
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TATA box in eukaryotic genes
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diff # of promotors which give the percent occurence of various bases at the positions that they indicate.
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metallothionein gene possesses several constitutive elements in its promotor...
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as well as specific response elements MRE and GRE. BLE involve basal level expression. TRE is tumor response.
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transcription factors
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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 |
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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 |
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more RNA pol II
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CTD is essential, but ISNT phosphorylated and can intiate transcription
TATA box (TATAAA) is promotor 7 Tf required |
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transcription initiation
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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) |
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nucleosome structure
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acK:acetylated lysine residue
meK:methylated lysine residue PS:P-ylated serine residue many modifiable sites and diff positions of aa |
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transcriptional regulation of eukaryotic genes
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DNA wrapped around nucleosomes
Tf bound to enhancer or silencer RNA pol II & GTF bound at promotor specific Tf's stimulate and repress transcription |
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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) |
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alpha helices and DNA
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helices fit into major groove of B-form DNA
diameter of helix=1.2nm (same as groove) |
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HTH motif
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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 |
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more HTH motif
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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 |
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HTH domains in sequence-specific DNA binding proteins
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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.
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Zn-finger motif
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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 |
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Zn-finger diagram
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coordination of Cys and His residues to Zn is horseshoe
secondary structure is ribboned and then straight |
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more Zn-fingers
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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) |
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leucine zipper motif
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in almost all organisms
28-residue, w/ leu every 7th and a basic region amphipathic a-helix and coiled-coil dimer |
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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 |
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model for dimeric bZIp protein
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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
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model for heterodimeric bZIP Tf c-Fos
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c-Jun bound to a DNA oligomer with the AP-1 target sequence TGACTCA
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post-transcriptional processing of mRNA in eukaryotes
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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 |
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eukaryotic genes are split
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introns bw exons
exons are 45-549 bases introns are spliced |
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capping and methylation
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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 |
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methylation of specific sites at 5' of euk pre-mRNA is essential in mRNA maturation
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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 |
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3'-polyadenylylation
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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 |
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polyA addition and cleavage diagram
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adds to 3' 10-35 nucleotides downstream from AAUAAA. cleavage and polyadenylylation makes a loop
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consensus sequences at the splice sites in vertebrae genes
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5' splice site
AG:GUAAGU exon intron 3' splice site Py Py..6x more-CAG:G-- intron exon |
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branch site and lariat
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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 |
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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 |
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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 |
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assembly of the spliceosome
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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 |
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events in spliceosome assembly
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U1 snRNP binds at 5' then U2 at branch point. U4 is released and lariat forms freeing the 3'&5'.
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