Exam 3 study guide break down Flashcards
3 primary mechanisms to find bacterial RNAP promoter
sliding
intersegment transfer
intradomain association association and disassociation (hopping)
when is holoenzyme in a closed complex
when it slides onto a promoter
when is holoenzyme in open complex
when sigma factor binds strongly
what happens when holoenzyme is in an open complex
DNA duplex is bent
promoter is denatured
transcription bubble gets bigger
jaws close around downstream sequence
what is abortivive initiation transcripts
RNAP pulls in DNA via a scrunching motion and the stress of that causes short abortive transcripts to come out
what happens from successive abortive initiation events
RNAP breaks away from promoter to transition to elongation
nonspecific interaction by sigma and DNA duplex
is when sigma interacts with the phosphodiester backbone in the closed binary RNAP complex
specific interaction between sigma and DNA duplex
sigma base pairs to -10 and discriminator facilitates the melting that leads to irreversible transition to open RNAP complex
positioning factors with TBP
TFIIIB
SL1
TFIID
where does TBP bind
minor groove in DNA
what happens when TBP binds
DNA bends 80 degrees
brings transcription factors bound upstream in close proximity with RNAP bound downstream
TATA containing initiation mechanism
TBP in TFIID directs TFs to TATA box
B binds
F binds
RNAPII binds
B binds to RNAP to assist D
H binds and phosphorylates CTD
E binds
complex formed
TATA less differences
TFIID binds to Inr via interactions with TAFs
some lack unique transcription start sites
what modification happens on the CTD of RNAPII
phosphorylation by TFIIH and cdk9
what happens when CTD is modified
promoter and transcription factor release
RNAP conformational change
disengages from general TF
tightens interactions with DNA
acquired new proteins to increase RNAPII processivity
recognition site for capping, tailing and splicing
how do elongation factors facilitate continued transcription
- recruit chromatin remodeling complexes to release chromatin that is blocking movement
- interacts with RNAP via a coactivator to unpause enzyme
- act as or recruit elongation factors
- decrease the likelihood RNAP will dissociate
- help RNAP move through nucleosomes
intron definition
5’ and 3’ splice sites are simultaneously recognized by components of the E complex.
U1 and then U2Af
used for small single intron genes in unicellular eukaryotes
exon definition
U2Af binds to 3’ splice site (end of first exon)
U1 binds to 5’ splice site (start of second exon)
complexes are switches to link across
used when introns are long and exons are short
U246 in splicesome interaction
U2 binds to branch point
U5 and U4/U6 bind
U1 and U4 are released
U6 binds to U2
U6 binding to U2 brings 5’ splice site close to branch point
RNAP termination allosteric changes
binding of cleavage factors and subsequent RNA cleavage leads to conformational change
makes enzyme more likely to dissociate
exonuclease tornado
RNA cleavage products produces an uncapped 5’ end which is bound by an exonuclease
exonuclease moves up and destroys DNA-RNA hybrid
RNAP dissociates
RNAP termination and tail formation
cleavage factors cleave the RNA via an endonucleolytic cut and then the tail is added
5’ to 3’ decay pathway
digest polyA tail to 10-12 nucleotides
Lsm1-7 decapping enhancer binds to short tail
Lsm1-7 activates decapping on 5’ end
removal of cap produces a 5’ monophosphorylated RNA
5’ to 3’ Xrn1 exonuclease goes to town
3’ to 5’ decay pathway
digestion of poly A tail to 10-12 nucleotides triggers exosome action
exosome has 3’to 5’ exonuclease
exome goes to town from 3’ end
tRNA charging by aminoacyl-tRNA synthetase
- Amino acids react with ATP to form aminoacyl adenylate intermediate
- 2’OH or 3’OH of the terminal 3’ nucleotide in the tRNA attacks the carbonyl carbon of the adenylate
- aminoacyl-tRNA and AMP are made
detecting tRNA differences
changes in nucleotide sequences
subtle differences in shape
direct (nucleotides)
indirect(phosphodiester)
common - anticodon loop and amino acid acceptor arm
detecting amino acid differences
shape of amino acids
different binding efficiencies and free energies
kinetic proofreading
tRNAs that match the specific nucleotide sequence combination for the synthetase properly align the amino acid acceptor stem with ATP and amino acid in active site
triggers aminoacetylation reaction
chemical proofreading
pretransfer editing
posttransfer editing
pretransfer editing
incorrect amino-acylAMP is hydrolized after tRNA binding but before charging
posttransfer editing
amino acid is hydrolized from aminoacyl-tRNA after charging by using an editing active site
1st sieve
amino acids larger than correct amino acid will be excluded from synthetic site and loading will not occur
2nd sieve
amino acids smaller than the correct amino acid will fit into the editing site and will be hydrolyzed and removed
IF3
helps stabilize 30S subunit and helps it bind to the initiation site on mRNA
IF2
aids in binding the initiator tRNA to complex
IF1
binds to 30s subunit at the A site and prevents tRNAs from binding prematurely
scanning model
small subunit binds to 5’ cap and moves 5’to 3’ done the mRNA
melts some secondary structures
stops when it recognizes the start codon and flanking sequences -4 and +1 (Kozak)
steps of elongation
EF-Tu-GTP binds to aminoacyl-tRNA
anticodon end moves to A site
EF-Tu-GTP end binds to factor binding center
EF-Tu-GTP hydrolysis
aminoacyl end moves to face the P site tRNA
EF-Tu-GDP released
EF-Ts regenerates EF-Tu-GTP
peptidyl transferase creates peptide bond
hybrid state
EF-G-GTP binds
EF-G-GTP hydrolysis by factor binding site
EF-G-GDP unlocks ribosome and opens gates
EF-G-GDP binds to A site
A site anticodon pushed to P site
P site anticodon pushed to E site
small subunits rotates and loses affinity for EF-G-GDP
EF-G-GDP dissociates
EF-G released GDP and picks up GTP
repeat
proofreading of EF-Tu
EF-Tu-GTP only interacts with factor binding center if aminoacyl-tRNA fully enters the A site
incorrect tRNA will dissociate before GTP hydrolysis
23S rRNA
positions aminoacyl end of aminoacyl-tRNA near the end of the peptidyl-tRNA
places amino group on A-tRNA in close proximity to the carbonyl group on P-tRNA
hybrid state
the anticodons of the tRNAs remain in their pre-peptidyl transfer positions
3’end of A-site tRNA is now bound to polypeptide and prefers the P site
3’end of P site tRNA is deacetylated and prefers the E site
EF-G-GDP is needed to open gates
termination via release factors
termination codons recognized by RF1 and 2
RF3-GDP binds to ribosome
polypeptide released
RF3-GDP turns to RF3-GTP
RF 1 and 2 dissociate
RF3-GTP hydrolyzed by factor binding center
RF3-GDP released
RRF binds to A site and recruits EF-G-GTP
EF-G-GTP hydrolyzed
unloaded tRNAs are released
EF-G-GDP, RRF, and mRNA dissociate
IF3 binds to dissociate ribosome subunits
lacOc
constitutive cis-acting mutation
repressor cannot bind to mutant operator
always on
lacI-
constitutive trans-acting mutation
defective repressor cannot bind
always on
LacIs
uninducible trans-acting mutation
super repressor
always off
lacI-d
dominant negative trans-mutation
one mutant subunit makes repressor nonfunctional
how does the repressor bind to the operator
inverted repeats in the operator, in major groove of DNA
when repressor binds to two operators
it induces a loop into the DNA between the two binding sites
what does repressor binding do to RNAP
repressor binding enhances RNAP binding to promoter
how often does active repressor bind to operator
96% of the time
how often does inactive repressor bind to operator
3% of the time
active repressor has a … affinity for random DNA
low
