Final Review Block 2 Flashcards
6 ways that prokaryotic transcription differs from eukaryotic transcription
- in the cytoplasm (not nucleus)
- no histones
- transcription and translation at the same time
- operons
- polycistronic
- attenuation
what is the role of sigma factor?
present in prokaryotes, unites RNA polymerase and DNA promoter sequences, responsible for unwinding DNA strands, released after 10 nt added
rho independent termination
GC rich hairpin followed by series of Us, the series of Us weakness the assoc between RNA pol and DNA and RNA pol falls off
rho-dependent termination
weak hairpin that is NOT followed by Us is present, this slows the RNA pol down, the rho protein associates and removes RNA pol from DNA
what is encoded for by the trp operon?
5 structural enzymes that form tryptophan synthetase
what occurs when trp is high?
trp binds to the repressor protein, repressor protein is activated and binds promoter/shielding in from RNA pol
describe attenuation
premature stop mechanism using “leader sequence”, with hairpins 1-2, 3-4
describe attenuation when trp is high
when trp is high, ribosome can add 2 trp without delay, 3-4 hairpin forms, rho independent termination occurs and ribosome falls off – premature termination
describe attenuation when trp is low
when trp is low, ribosome stalls waiting for 2 trp to add, it stalls over region 1 and 1-2 cannot form, but 2-3 forms instead, thus 3-4 cannot form and ribosome continues
what is controlled by the lac operon?
expression of LacZ protein- codes for enzymes that are used to digest lactose
describe (+) glucose, no lactose
operon is repressed, no LacZ
describe (+) glucose, (+) lactose
presence of lactose inhibits repressor, polymerase is able to bind, yields low expression of LacZ
describe no glucose, (+) lactose
catabolic activating protein present, no repressor, high LacZ expression
what is synthesized by the different RNA polymerases?
RNA pol I- 5.8s, 18s, 28s rRNA
RNA pol II- mRNA, sno/sn/miRNA
RNA pol III- tRNA, 5s rRNA
what is significant about the -35 and -25 sites in eukaryotic transcription?
- 35- GC rich recognition site
- 25- TATA box
what are the steps in formation of the pre-recognition complex?
DBFEH
dogs bark and bite people frequently every halloween
action of TFIID
bind TBP and together they bind TATA box
action of TFIIH
acts as helices, phosphorylates 5th serine on CTD on RNA pol II leading to release of transcription factors
describe RNA pol I initiation and termination
initiation- upstream binding factor, SL1 (TBP+TFs)
termination- rho dependent-like
describe RNA pol III initiation and termination
initiaiton- ses internal control regions (except U6 snRNA)
termination- rho independent-like
where is mRNA processed?
in the nucleus
describe addition of 5’cap
occurs when mRNA about 25 nt long (during transcription), 5’triphosphate is removed by phosphohydrolase, GTP is added in unique 5’-5’ fashion by guanylyl transferase, methyl groups are added
3 functions of 5’cap
prevent exonuclease activity
for positioning on ribosome
to delineate 5’ end for splicing
describe addition of poly-A tail
cleavage proteins and PAP detect and bind to AAUAAA and GU motif at 3’ end, complex cleaves 10-35 nt, PAP adds 12 A slow, PABPII adds 200-250 As fast
2 functions of poly-A tail
prevent exonuclease activity at 3’end, recognition signal for mRNA transport into cytoplasm
what composes the sliceosome?
snRNA (U1,U2,U4, U5,U6) + splicing factor proteins
what are the conserved sequences for splicing
5’ GU
branch point- A
3’ AG
3 steps of splicing
1-adenine residue 2’OH attacks phosphodiester bond at 5’splice site
2-3’OH on exon is exposed, attack 3’ splice spot
3- intronis removed in “lariat” shape and exons are fused together
what is eukaryotic splicing similar to?
group 2 self splicing- mito of fungi, chloroplasts and cyanobacteria, also have lariat byproduct
clinical correlation to splicing
b-thalassemia- mutation oin b-globulin gene leads to alteration in splice sits and abnormal product (longer exon 3)
A-I editing
Adenosine changed to Inosine (like guanine)
enzyme is ADAR
C-U editing
cytosine to uracil
enzyme is cytidine deaminase
clinical example of C-U editing
in gut, C-U editing yeilds shorter ApoB product (changes from glutamine to stop codon)
editing in pathogenic trypanosomes
extensive editing
uracil edits by uridylyl transferase and guide RNAs
what is responsible for rRNA edition?
snoRNA
what is the action of snoRNA?
will cleave 45s into 5.8s, 18s, 28s units, can also directly methylate and pseudouridylate
what are the 4 ways in which tRNAs are edited?
cleavage- 3’ end by RNase D, 5’ end by RNase P
splicing- facilitated by proteins
RNA editing- uracil at 3’end changed to CAA
chemical modifications by snoRNA
what is the significant of 3’CAA in tRNA?
responsible for charging tRNA with aa
what is meant by the genetic code is “degenerate”?
61 codons for only 20 aa (repeats)
what are the start and stop codons?
start- AUG
stop- UGA, UAG, UAA
what aa only have 1 codon?
Met and Trp
what enzyme will “charge” tRNAs? how many are there?
aminoacyl tRNA synthetase, 20- one for each aa
what is the wobble position?
3rd nt on mRNA, 1st nt on tRNA, allows for non-standaed base pairing
describe prokaryotic ribosome
large= 50s (23s + 5s + 31 proteins) small = 30s (16s + 21 proteins) overall = 70s
describe eukaryotic ribosome
large=60s (28s+5.8s+50 proteins)
small= 40s (18s+33 proteins)
overall=80s
what are the 3 sites on a ribosome?
A site- aminoacyl
P site- peptidyl
E site- exit
differentiate between the 2 tRNAs for Met
one can only initiate protein synthesis, the other is for incorporation of Met into a growing chain; both have same aminoacyl synthetase
where does tRNAi-Met bind?
binds to the P site, all other tRNAs bind the A site
describe DNA methylation
methyl groups added to cytosines in CpG islands located within the gene promoter region by DNA methyltransferase
what is the effect of hyper vs. hypo methylation?
hyper- silencing
hypo-activating
how does methylation silence gene expression?
1-makes it harder for transcription factors to bind
2- recruits methyl binding proteins that recruit enzymes that modify chromatin and make it hard for transcription factors to bind
rett syndrome
caused by dysfunction of MECP2- methyl binding protein
what is the overall effect of histone acetylation? why?
activation, acetylation of histones will shield DNA from (+) lysine and will decrease the attraction between DNA and histone, decreased interaction = looser DNA = increase access
what enzyme is responsible for histone acetylation?
histone acetyltransferases
describe bromodomains
found on transcription factor proteins, able to bind acetylated lysine’s
what is the effect of histone methylation?
activating or silencing
H3K9
when methylated- decreased gene expression, will recruit HP1, leads to more methylation
when acetylated- will increase gene expression
H3K4
when methylated, will increase gene expression
describe the actions of nucleosome remodeling engines?
use ATP to shift the position of nucleosomes that will result in varied access to gene promoters
where are repressors and activators working?
at the promoter proximal region (-200 to -100), both can directly bind DNA
what is the action of corepressors/coactivators?
bind to transcription factors, NOT directly to DNA
what works at a distance to influence expression?
enhancers- can be 200 bp to 5kbp up or down stream. allow for synergistic binding of transcription factors
example of coactivators
scaffold proteins
example of corepressors
histone deacetylase - removes acetyl groups and can also recruit methyltransferases to methylate
describe translational control in times of low iron
increase receptor: IRE-BP binds to AU rich hairpin in 3’UTR to protect from endonucleases
decrease ferritin: IRE-BP stablizes 5’ hairpin and ribosome cannot start translation
describe translational control in times of high iron
increase ferritin: no IRE-BP, ribosome translates ferritin
decrease receptor: no IRE-BP, mRNA is degraded by endonucleases
describe siRNA in the 3’UTR
exogenous
perfect mRNA binding
results in mRNA cleavage
describe micorRNA in the 3’UTR
endogenous (from RNA pol II)
imperfect mRNA binding
results in translational repression or mRNA degradation
describe the nuclear localization signal
basic aa sequence
describe the 5 steps in nuclear import
- NLS assoc with import in
- (p+importin) travel through NPC via hydrophobic interactions with FC nucleoporins
- (P+importin) bind GTP and protein is released
- (GTP+importin) exit through NPC
- in cytoplasm, GTP interacts with GAP, and is converted to GDP, releasing importin
what is GAP?
GTP –> GDP in cytoplasm
what is GEF?
exchanges GTP for GDP in nucleus
describe the nuclear export signal
hydrophobic with leucine residues
describe the 3 steps in nuclear export
1-(p+exportin+GTP)pass through NPC
2- GTP interacts with GAP in cytoplasm and is changed to GDP, thereby releasing protein and exportin
3- exportin assoc with GDP and travels back into nucleus
describe the mitochondrial targeting sequence
amipathic a-helix with hydrophobic residues on one side and basic residues on the other side
what is unique about mito import?
proteins must be unfolded to enter mito, thus they are accompanied by chaperones
describe contact site on mito membrane
contact sites exist between inner and outer membranes so that proteins are transported directly into mito matrix
what sequence targets for ER?
signal sequence at N-terminus- (+) charge followed by hydrophobic residues
where do proteins end up if they are inside of ER lumen?
outside of the cell
what allows entry into ER lumen?
translocons
describe type I integral membrane proteins
have COO- in cytoplasm
where is the signal sequence in type I integral membrane proteins?
at N-terminus
what is the significance of STA sequence?
Stop-Transfer-Anchor sequence will span the membrane, closes the translocon so that the remaining portion of the protein (COO- end) will be in cytoplasm
describe type II integral membrane proteins
have NH3+ in cytoplasm
what types of sequences are found in type II integral membrane proteins?
Signal sequence- bind and opens translocon
anchor sequence- spans membrane
*No stop sequences- translation continues into ER lumen
what targets to COPI vesicles?
KDEL
what targets to lysosome vis clathrin?
mannose-6-phosphate
what targets to COPII?
diacidic motif
what is the activity of COPI vesicles?
retrograde transport from golgi to ER
what is the activity of clarthin vesicles?
incoming from cell surface or to lysosomes, dynamic help with “pinching”
southern blot, probe?
used to assess DNA with nucleic acid probe
PCR
for DNA quantification, specificity convey via primers
sequencing
melt DNA, add primer and modified bases
actual DNA is complimentary to what is found experimentally
northern blot, probe?
RNA with nucleic acid probe
must denature to ensure linearity, use formaldehyde probe
RT-PCR
for mRNA, use RT to make cDNA
western blot
use to assess protein levels with antibody probe
mutation that causes CF
mutation in CFTR leading to faulty Cl channel
what can be used to treat class I CF?
gentamycin by suppressing recognition of premature stop codon
what class is the mc/ cause of CF?
class II- DF508, within NBD1 domain, leads to increased levels of misfolded proteins, increased ubiquitination and increased turnover at cell surface
describe folding of wild type CFTR
up to 50% wild type CFTR is misfolded, chaperones in ER will help. if incorrectly folded, will lack diacidic motif and will not be incorporated into COPII vesicles
2 classes of DF508 treatment
correctors- enhance folding/trafficking to membrane
potentiatiors- decrease turnover and increase activity of channel
testing for CF
prenatal- multiplex PCR assay or NextGen sequencing
post-natal- sweat test
