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MCB - Molecular Bio > Eukaryotic Transcription and Post-Transcriptional Regulation > Flashcards

Eukaryotic Transcription and Post-Transcriptional Regulation Flashcards

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Biology 101 flashcards
1
Q

central dogma

A

DNA to RNA to protein

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2
Q

gene regulation

A 
chromatin mods (histone mod, acetylation/methylation)
transcriptional control by TFs/Pol
RNA processing control
RNA transport and localisation control
mRNA degradation
translational control
protein activity control
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3
Q

transcriptional control

A

chromatin structure
Pol binding
activation factors
additional binding

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4
Q

chromatin structure modifications

A

highly packed heterochromatin not expressed

acetylation: acetyl to +ve lysines in histone tails loosens chromatin
methylation: condense chromatin
phosphorylation: phosphate next to methylated AA loosen chromatin

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5
Q

histone code hypothesis

A

chemical mods to histones and DNA determine chromatin configuration so transcription

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6
Q

chromatin can move within nucleus to…

A

alter gene expression
active are central
heterochromatin is close to membrane

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7
Q

RNA polymerase binding for transcriptional control

A

3 types: RNA Pol I ribosomal RNA gene
RNA Pol II protein coding small RNAs
RNA Pol III tRNA rRNA some snRNA so other small RNAs

initiation complex

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8
Q

TATA box (prokaryotes have TATAAT at -10)

A

consensus sequence
in pol II promoters
-25 to transcriptional start

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9
Q

RNA Pol II initiation complex

A

pol with TFs fine tune process and stabilise and activate pol

TFIID: TBP (TATA box binding protein) and TAF (regulates DNA binding of pol)

TFIIB: positions RNA pol at start site

TFIIF: stabilises RNA Pol

TFIIE: attracts TFIIH

TFIIH: unwinds DNA and phosphorylates ser5 (in CTD tail of pol II)

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10
Q

TBP

A

binds TATA box and bends DNA so RNA/TFs can bind and stabilised by TFIIF and E and H help binding

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11
Q

additional binding and activation factors (activator proteins, TFs, proximal control elements and distal, combinatorial)

A

activator proteins bind to enhancers in promoter region so form mediator complex, sends info from promoter to RNA Pol and tells Pol to transcribe
(some promoters so far from start so needs to bend by DNA-bending proteins)

TFs initiate transcription and help RNA pol

proximal control elements close to TATA while distal enhancers are far away

combinatorial control: comb of control elements active when appropriate activators are present

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12
Q

TFs (e.g.)

A

contain DNA binding domains

Leucine zipper TF bind to promoters and cause transcription

recognise specific features of DNA

Zinc finger TF bind promoter

TFs work together

can also inhibit transcription

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13
Q

forward genetics

A

identify gene (function) from phenotype e.g. moles, red skin, skin damage is XPD gene functions in DNA repair

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14
Q

reverse genetics

A

predict phenotype from gene analysis

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15
Q

analysing gene expression

A

single gene: RT-PCR, live cell imaging, promoter studies

all genes: microarrays, RNA sequencing

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16
Q

RT-PCR

A

cDNA to PCR to electrophoresis

1 gene at a time

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17
Q

live cell imaging

A

microscope analyse gene activity and localisation
shows why gene expressed
can fuse to reporter gene (GFP) to see where mRNA expressed in cell

18
Q

promoter analysis

A

to see which enhancer motifs affect transcription

add reporter gene to promoter and transfect to cells and see green colour where expressed

19
Q

ChIP (chromatin immunoprecipitation)

A

Abs for TFs so pull out sequence with TF so find where it binds and see if changes from disease/env
or amplify sequence and gap is where TF binds

20
Q

phylogenetic fingerprinting

A

find sequence where TF binds

21
Q

microarrays

A

study expression of interacting groups of genes
automated
compare patterns
microscrope slide with mRNA hybridised to labelled cDNA so show expression levels
1 spot means 1 gene and darker colour means more gene expressed

22
Q

RNA sequencing

A

show which exons transcribed in a sample
count number of RNAs per exon in sample so calculate expression level of a gene
also identify gene splicing variants

23
Q

what is the point of post-transcriptional regulation of gene expression?

A

for mRNA stability, translation, protein function

24
Q

types of processing

A

capping at 5’ end
poly-A tail to 3’
splicing to remove introns

25
Q

RNA Pol II and regulation

A

makes mRNA
CTD tail - long with loops, 4 serines at position 5 is where phosphorylation happens

TFIIH is involved in phosphorylation
RNA Pol onto DNA and transcribes it - tail already phosphorylated on serine 5, processing factors can attach to tail and hop to pre-mRNA

26
Q

capping

A

makes mRNA stable and protects from enzymes and important for export and translation

5’ pppNpNp (phosphate and any base)
phosphatase removes 1 phosphate so only 2 p’s at start
G added to this end by guanyl transferase then G methylated
another methylation occurs of the downstream base

27
Q

poly-A

A

3’ end polyadenylation

3’ sequence: AAUAAA-CA-GU rich region

  1. 3’ sequence removed by cleavage factor but need to cleave at correct position so don’t remove important nucleotides

CPSF (cleavage and polya specificity factor) binds AAUAAA

G/U rich regions is in part meant to be cleaved and CstF (cleavage stimulation factor F) recognises it

CFII (cleavage factor) recognise CA, bend it so PAP (poly A pol) can bind and chop off end tail

polyA factor waits will AAUAAA then PAP adds As with ATP and CstF/CFI/CFII removed

PABP (poly A binding protein) binds to A tail and helps add As faster

28
Q

introns

A

remove and ligate 2 exons

5’ end splice site and 3’ end splice site
‘A’ branch point in intron where join to form lariat

2 step transesterification

  1. hydroxyl group at branch point A that binds to phosphate group causes attached lariat to 3’ exon
  2. loops removed and 2 exons ligated with esterification
29
Q

snRNPs

A

small nuclear ribonucleoprotein particles

in spliceosome and 200 other proteins involved

30
Q

specificity (introns exons)

A

sequence at exon/intron junction at 5’ end 3’ end and in middle
so splicing gives specificity

31
Q

splicing

A

5’ exon/intron junction sequence recognised by U1 snRNP
BBP (branch point binding protein) binds to branch point A
which is joined by U2AF (U2 auxilliary factor) which helps U2 snRNP find A
U4/U6 binds to U1+U2 so conformational change and 1st cut and transesterification forms loop
U4+U1 no longer required
U6 causes 3’ intron/exon cleavage and exons fused afterwards

32
Q

snRNPs give specificity in splicing

A

U1 has RNA sequence matching 5’ exon/intron junction

cells don’t rely on snRNPs for splicing because exon length same ish but intron size changes so can’t cut on basis of distance
but conserved in some organism so more accuracy to splicing

33
Q

ESEs

A

exonic splicing enhancer

SR protein binds and shield exon

34
Q

hnRNP

A

heterogeneous nuclear ribonucleoprotein

bind introns and rip up so branch point found so A branchpoint can reach 5’ exon/intron junction
(because introns quite long sometimes)

35
Q

alternative splicing

A

evolution, new mRNAs for new function

recognise new parts maybe

36
Q

RNA editing (definition, types, why)

A

alter sequence of pre-mRNAs (not splicing or methylation) after transcription

base insertions
cytosine deamination to uracil
adenine deamination to inosine
(examples lecture 15)

to revise mistakes, plasticity for function, defense)

37
Q

base insertions

A

usually uracil
sites missing U specified by guide RNA1
edits by pairing to guide RNA2

e.g. sleeping sickness

38
Q

cytosine deamination to uracil

A

AA change so new protein

e.g. apoliprotein B

39
Q

adenine deamination to inosine

A

protein change by ADAR (adenosine deaminase acting on RNA)

e.g. glutamate receptor

40
Q

ribozymes

A

catalytic RNAs bind to mRNA to cleave and destroy

intron RNA can splice w/o spliceosome
RNAse P ribonuclease in processing tRNA and small RNAs, peptidyl transferase ribozyme of ribosome
in viruses as well
rRNAs of ribosomes

can be therapy for cancer

41
Q

regulation of nuclear export of mRNAs

A

only processed mRNA goes through pore to cytosol because pores restrict movement of molecules
needs energy to guide through pore
need export or would be degraded

42
Q

miRNA

A

small RNAs in junk part of DNA, for regulation of transcription, defence, inhibit/destroy transcripts

21-25 nucleotides, non-coding, single strand, matching mRNA so targets specific

RISC complex helps (RNA-induced silencing complex)
Pol II in miRNA production causes pri to pre to mature single small RNA and cropping dicing so 1 strand digested and 1 to RISC

MCB - Molecular Bio (7 decks)

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