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BIO230 > Lecture 5 and 6 > Flashcards

Lecture 5 and 6 Flashcards

1
Q

How does transcriptome analyses provide a signature of cell state?

A

It shows response to extracellular stimuli and disease states (eg. cancer)

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2
Q

Are translation and transcription coupled in bacteria?

A

yes

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2
Q

Technique that allows for Transcriptome analysis

A

DNA microarray

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2
Q

IC50

A

Concentration of compound required to inhibit cell proliferation by 50%

lower IC50 is stronger

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3
Q

How is eukaryotic RNA processing tightly coupled to transcription?

A

Due to covalent modifications of RNA ends and removal of intron sequences.

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4
Q

RNA capping

A

Addition of a modified guanine nucleotide to the 5’ end of pre mRNA

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5
Q

What is the RNA cap bound by?

A

Cap binding complex (CBC)

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6
Q

5’ exonuclease/5’ to 3’ exonuclease

A

They cut phosphodiester bonds in the 5’ to 3’ direction.
The 5’ cap helps protect from this.

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7
Q

Functions of 5’ cap

A
  1. helps in RNA processing and export from the nucleus.
  2. Important role in translation of mRNAs in the cytosol.
  3. Protects mRNA from degradation.
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8
Q

Coding sequences

A

exons

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9
Q

Non-coding sequences

A

Introns

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10
Q

RNA splicing

A

both exons and introns are transcribed into RNA.
the removal of introns from RNA is called RNA splicing.

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11
Q

Spliceosome

A

Enzyme complex made of RNA and proteins that carries out RNA splicing

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11
Q

Alternative splicing

A

Different cells splice RNA transcript differently to make different proteins from the same gene.

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12
Q

What percentage of human genes produce multiple proteins and why?

A

75%
It increases coding potential of genomes

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13
Q

Exon Junction complexes (EJCs)

A

Sites of proper RNA splicing are bound by EJCs, They serve as a marker for properly spliced RNA.

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13
Q

Alternative splicing regulation

A

Negative control: When no repressor splicing occurs, when repressor present splicing does not occur.

Positive control: When activator present splicing occurs, when n activator present splicing does not occur.

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14
Q

Example where alternative splicing regulation takes place

A

Drosophilia sex determination

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15
Q

How is Drosophilia sex determines?

A

ratio of X chromosomes: autosomal sets

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16
Q

X:A=0.5
X:A=<0.5

A

male
metamale

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17
Q

X:A=1
X:A=>1

A

female
metafemale

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18
Q

X:A between 0.5 and 1

A

Intersex

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19
Q

Three genes involved in Drosophilia sex determination

A

Sex Lethal, Transformer and Doublesex

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20
Q

Sex Lethal

A

Splicing repressor

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21
Q

Transformer

A

Splicing activator

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22
Q

Sex determination in male drosophila - regulation

A

not regulated by sex lethal (not spliced) and transformer (spliced).
doublesex protein represses female gene expression.

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23
Q

Doublesex

A

Regulates sex gene expression

24
Q

Sex determination in female drosophila - regulation

A
  1. The splice site is blocked
  2. Functional Sex-lethal protein is produced.
  3. Sex-Lethal blocks splicing of Sxl and TRa.
  4. Functional Tra protein is produced.
  5. Tra activates splicing of doublesex.
  6. Dsx protein will repress male gene expression.
  7. Female development.
25
Q

3’ polyadenylation

A
  1. More complex than transcription termination is prokaryotes
  2. Signals encoded in genome
  3. RNA polymerase transfers protein complexes to RNA
26
Q

What protein complexes does RNA polymerase transfer to RNA?

A

CstF (Cleavage stimulating factor)
CPSF (cleavage and polyadenylation stimulating factor)

27
Q

Review of coupling of Transcription and RNA Processing

A

During transcription elongation, the C-terminal (CTD) of RNA polymerase binds RNA processing proteins and transfers them to RNA at the appropriate time.

27
Q

Review of RNA processing - 3’ polyadenylation

A
  1. RNA is cleaved
  2. Transcription terminates
  3. Poly-A-Polymerase adds ~200 A nucleotides to the 3’ end of the RNA from ATP (not genome encoded)
  4. the poly A tail is bound by poly A binding proteins.
27
Q

What are the markers of mature mRNA?

A

cap binding complex, exon junction complexes, and poly-A-binding proteins.
They must be acquired for export

28
Q

What does the poly-A-tail aid in?

A

RNA export, translation and mRNA stability

29
Q

How is the binding of RNA processing proteins to RNA regulated?

A

By phosphorylation of RNA polymerase

30
Q

What degrades improperly processed mRNAs? Where are improperly processed mRNAs degraded?

A

exosome, in the nucleus

30
Q

What are the markers of immature mRNA?

A

Proteins involved in RNA splicing (snRNPs)
they must be lost for export

31
Q

Review of translation

A
  1. tRNAs match amino acids to codons in the mRNA genetic code
  2. mRNA message is decoded in ribosomes made up of >50 different proteins and several RNA molecules.
  3. Amino acids are added to the C-terminal end of the growing polypeptide chain.
32
Q

In what direction are proteins synthesised?

A

From N to C terminus.

33
Q

mRNA quality control in eukaryotes

A

the eukaryotic translation initiation machinery recognizes the 5’cap and poly-A tail.
Eukaryotic initiation factors: eIF4E and eIF4G will recruit small ribosomal complex which will initiate translation at first AUG
EJC also stimulates translation ensuring proper splicing

34
Q

eIF4E

A

binds 5’ cap

35
Q

eIF4G

A

binds poly-A-binding protein

36
Q

Nonsense-mediated mRNA decay

A
  1. prominent mRNA surveillance system
  2. surveys for nonsense (STOP) codons in the wrong place
  3. indicator of improper splicing
37
Q

Upf proteins

A

They are protein that will trigger mRNA degradation whenimproper splicing has occurred and a stop codon is still present in the mRNA

38
Q

Normal splicing

A
  1. The ribosome binds mRNA as it emerges from the nuclear pore
  2. EJCs are displaced by the moving ribosome, they also make protein
  3. The stop codon is the last codon
  4. no EJCs remain bound when the ribosome reaches the stop codon.
  5. mRNA is released in the cytosol.
39
Q

Abnormal splicing

A
  1. The ribosome binds mRNA as it emerges form the nuclear pore.
  2. EJS are displaced by the moving ribosome.
  3. the stop codon is premature.
  4. The EJCs remain on the mRNA when the ribosome reaches the stop codon.
  5. mRNA is degraded with the help of Upf proteins.
39
Q

functions of Nonsense-mediated mRNA decay

A
  1. evolution of eukaryotes - allowing selection of DNA rearrangements or alternative splicing patterns
  2. role in cells of the immune system where extensive DNA rearrangements occur to produce antibodies.
  3. helps degrade aberrant mRNA and allows functional protein to accumulate.
40
Q

Prokaryotic quality control for mRNA

A
  1. Ribosomes stall on broken or incomplete mRNAs and do not release
  2. A special RNA tmRNA is recruited to the A site
  3. It carries an alanine amino acid
  4. It acts as both a tRNA and an mRNA
  5. Broken mRNA is released.
  6. Alanine is added onto the polypeptide from the tmRNA , which acts like a tRNA but with no anticodon-codon binding.
  7. the ribosome translates 10 codons from the tmRA which now acts as an mRNA
  8. the 11th amino acid tag is recognized by proteases that degrade the entire protein.
41
Q

deadenylase

A

type of exonuclease that involves poly-A-shortening. it also binds the 5’ cap

42
Q

mRNA degradation in eukaryotes

A

Once the poly-A-tail reaches a critical length (human=25 nucltoides) two degradation mechanisms can occur:
1. Decapping followed by rapid 5’ to 3’ degradation
2. continued 3’ to 5’ degradation
Sometimes cytoplasmic poly-A elongation can also occur to stabilize mRNA

43
Q

Transferrin receptor

A

imports iron into the cell, needed when cellular iron Is low

43
Q

aconitase

A

example of protein that interferes with poly-A-shortening

44
Q

What happens when there is iron starvation?

A
  1. mRNA stabilized by cystolic aconitase
  2. it binds to 3’ UTR
  3. mRNA is stable and translated
  4. transferrin receptor made
45
Q

What happens when there is excess iron?

A
  1. Aconitase binds iron and undergoes a conformational change.
  2. mRNA is released and 3’UTR endonucleolytic cleavage site is exposed (polyA is removed); mRNA is degraded
  3. no transferrin receptor made
45
Q

miRNA

A
  1. regulate mRNA stability
  2. they base pair with specific mRNAs
  3. synthesized by RNA polymerase II and get a 5’cap and poly-A-tail
  4. after special processing miRNA associates with a protein complex called RNA induced silencing complex (RISC)
45
Q

extensive match of mRNA with miRNA

A

one strand already is degraded
then, miRNA will bind to mRNA
Slicing will take place
ATP is converted to ADP
RISC released for reuse and mRNA is degraded.

46
Q

What does miRNA associate with?

A

RNA induced silencing complex

46
Q

less extensive match of mRNA with miRNA

A

one strand already degraded
then, there is rapid translational repression, deadenylation and in most cases eventual degradation of mRNA

46
Q

important prroteiin RISC

A

A protein of RISC is argonaute, which plays a specific role in base pairing miRNA with mRNA

46
Q

RNA interference (RNAi)

A
  1. Double stranded RNAs that end up supressing the gene expression of other RNAs in a sequence-specific manner
  2. the protein used in the miRNA regulatory mechanism also serves as a defense mechanism against foreign RNA molecules
47
Q

Where is RNAi found?

A

eukaryotes, including fungi, plants and worms

48
Q

RITS

A
  1. interacts with newly transcribed RNA
  2. recruiter chromatin modifying enzymes
48
Q

CRISPR CAS Immunity - prokaryotic immunity

A
  1. short fragments of viral DNA integrate into the CRISPR region of the genome and become templates to produce crRNAs (CRISPR RNAs)
  2. Viral DNAs complementary to CRISPR reions are directed for degradation by Cas (CRISPR associated proteins)
48
Q

RNAi and siRNAs

A

RNAi destroys double stranded RNA
initiated by dicer protein complex which cuts RNA into siRNAs
siRNAs can interact with Arganaute and RISC proteins and follow the miRNA route to destroy double stranded RNA or they can also regulate transcription

48
Q

how do siRNAs regulate transcription

A

siRNAs interact withand RNA induced trnascriptional silencing (RITS) complex

BIO230 (6 decks)

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