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Molecular Biology Of The Gene > Post Transcriptnsl Gene Expression And Regulation > Flashcards

Post Transcriptnsl Gene Expression And Regulation Flashcards

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1
Q

What happens to mRNA after transcription?

A

5’ end capping = addition of CAP structure
3’ end processing = addition of PolyA tail/polyadenylation
Splicing = intron removal and joining of exons

These events happen co-transcriptionally (these changes happen at the same time RNA is being transcribed)

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2
Q

What is the 5’ CAP and what are the biological roles of the 5’ end CAP?

A

The 5’ cap is 7-methylguanosine

  • mRNA stability: protects RNA from degradation
  • AIDS with splicing and polyadenylation processes
  • translation initiation (CBC replaced by translation initiation factor)
  • in the nucleus cap is bound by cap-binding complex (CBC) which facilitates the export of the transcript from the nucleus
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3
Q

What are the biological roles of the 3’ end PolyA tail?

A

mRNA stability: protects RNA from degradation

Translation efficiency

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4
Q

What are the biological roles of splicing?

A

Source of genetic variation and complexity

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5
Q

What is the carboxy-terminal domain (CTD) of RNA polymerase II?

A

On the tail of the CTD of the RNA Polymerase II, there is a repeat of amino acids that contains specific serines that can be phosphorylated. Depending on what serine residue is phosphorylated determines what complex the RNA Polymerase II DNA interact with.

When the CTD is unphosphorylated it allows the assembly of the pre-initiation complex at the promoter. Promoter clearance happens when CTD is phosphorylated.

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6
Q

What does the CTD regulate?

A

Promoter clearance
Promoter escape and beginning of transcription
Different phosphorylation of the CTD allows the RNA polymerase to interact with different complexes;
- capping complex when serine number 5 is phosphorylated
- when serine number 2 is phosphorylated, polymerase is fully elongated and interacts with splicing machinery

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7
Q

What is the processing of capping?

A

CAP is added in a series of enzyme mediated steps. These enzymes associate together in the capping enzyme complex (CEC)
1) RNA triphosphatase cleaves the third phosphate from the 5’ end of RNA

2) RNA guanyltransferase adds GMP from GTP to RNA diphosphate
3) methylation of guanine by methyltransferase

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8
Q

What is polyadenylation?

A

Polyadenylation is the addition of a PolyA tail to the 3’ end of an mRNA molecule.
A PolyA tail is a series of adenosine monophosphates (stretch of RNA with only adenine bases)

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9
Q

How does polyadenylation take place?

A

Polyadenylation has been linked with the termination of transcription of the mRNA molecule

Cleavage of the pre-mRNA occurs between conserved AAUAAA (PolyA signal) and G/U sequence rich elements (downstream signal element), with these regions being separated by approximately 20 nucleotides.
These regions are recognised by cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF) respectively.
Cleavage occurs about 10-30 nucleotides after AAUAAA signal and the PolyA tail is added to the free 3’ end
The enzyme polyadenylate polymerase (PAP) catalysed the addition of 100-250 adenosines
PolyA binding protein (PABP) increases the affinity of PAP for RNA for further PolyA extension

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10
Q

What are CPSF and CstF?

A

CPSF is the cleavage and polyadenylation specificity factor: it cuts the pre-mRNA and interacts with the polyadenylate polymerase (PAP)

CstF is the cleavage stimulation factor

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11
Q

What is RNA editing?

A

RNA editing is the alteration of the open reading frame of an mRNA molecule which specifically changes the sequence of a small subset of transcripts, altering their sequence without affecting the sequence of their parents

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12
Q

What types of RNA editing are there?

A

Base modification - result sin an mRNA of same length but modified sequence

Insertion/deletions - results in a shorter or longer mRNA with a new open reading frame

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13
Q

What are base modifications in RNA editing?

A

Both base modifications are base deaminations (-NH2 replaced by O)

Adenosine (A) –> Inosine (I) by ADAR enzymes (adenosine deaminase that act on RNA)
,
Cytidine (C) –> Uridine (U) by CDAR enzymes (cytosine deaminase enzymes that act on RNA)

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14
Q

Why do you want to edit RNA?

A

Change protein sequence which contributes to protein variability

May induce a premature stop codon to get a truncated protein

May alter regulation: may change splicing or even binding of miRNAs and/or regulatory proteins

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15
Q

What is an example of RNA editing?

A

Apolipoprotein B (ApoB) is a plasma protein that plays a key role in the assembly, transport and metabolism of plasma lipoproteins.
There are two forms:
- Long form ApoB-100 which is synthesised by liver and experiences no editing

  • Short form ApoB-48 which is produced by small intestine
    Short form is RNA edited –> APOBEC1 is catalytic RNA specific cytosine deaminase which is bound to ACF which is an RNA binding protein that recognises a specific 11nt sequence –> deaminase a cytosine so a stop codon is generated resulting in a truncated protein
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16
Q

What is alternative polyadenylation (APA)?

A

The use of a different polyadenylation site to yield different mRNA isoforms
It can lead to changes in both coding sequence and 3’ UTR

Skipped terminal exon - exon is cleaved before the last exon
Composite terminal exon - include a bit of intron as splice site has been minced

3’ UTR region is the site of interaction of regulatory molecules. mRNA containing different 3’ UTR are regulated differently

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17
Q

What is RNA interference?

A

RNA interference is the repression of gene expression by RNA

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18
Q

What are the three classes of purposefully synthesised regulatory RNA?

A

Micro RNA (miRNA)

Piwi interacting RNAs (piRNA) - only found in germline cells of animals and repress the activity of transposons such as LINEs and SINEs

Long non-coding RNA (>200 nucleotides)

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19
Q

Describe miRNAs.

A

miRNAs are repressors (silencers) of gene expression

They are short RNAs (approximately 22bp) that are complementary to a sequence within target mRNAs

Present in all eukaryotes but absent in prokaryotes

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20
Q

How do miRNAs regulate gene expression?

A

When the complementarity of miRNA is perfect with target mRNA - it causes cleavage of target mRNA upon interaction or base pairing

When the complementarity of miRNA is not 100% perfect with mRNA - it reduces the stability of the target mRNA (causing it to be degraded)

Inhibits translation of target mRNA

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21
Q

Describe the biogenesis of miRNA?

A

derived from a primary miRNA transcript that includes an imperfectly paired stem-loop region

Microprocessor complex which contains dgcr8 (binds double stranded RNA) and an RNase known as drosha (has RNase activity and can cut RNA) excises the stem-loop from the rest of the molecule. Stem-loop is known as pre-miRNA

Pre-miRNA enters cytoplasm where RNase dicer works on it. Dicer removes loop leaving 22bp dsRNA molecule with short 3’ base overhangs

dsRNA gets incorporated into RISC complex and is unwound, with one strand retained in the RISC complex and the other discarded

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22
Q

What are three ways in which miRNAs can be produced?

A

Single miRNA gene

Clustered miRNA genes

Intron of protein-coding gene

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23
Q

What are the domains of the RISC complex?

A

PAZ domain - binds to end of dsRNA
Two RNAse III domains - cleave double stranded RNA

PAZ domain binds the end of dsRNA and because of the length of the turns of dsRNA, the loop of the precursor sites exactly at the right soar me of the RNase III domains

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24
Q

How does the miRNA bound RISC complex inhibit gene expression?

A

If there is high complementarity between the miRNA molecule in RISC and target mRNA, RISC complex will cleave the target molecule

If there is low complementarity between the miRNA molecule in RISC and target mRNA, RISC complex will deadenylate the target mRNA (ultimately leading to mRNA degradation)

The interaction between miRNA and target mRNA often happens in the 3’ UTR of the mRNA.

25
Q

Why are miRNAs biologically important?

A

1569 miRNAs have been found to date in humans (2017)
A single miRNA may be able to base-pair with numerous mRNAs and so regulate expression of many genes
When dicer is inhibited in developing oocytes, approximately 1/3 of all genes show increased expression

26
Q

What are short interfering RNAs (siRNA)?

A

siRNAs are similar to miRNAs but:

  • are not usually encoded purposefully by the genome
  • are probably involved in defence against unwanted RNAs

Long dsRNA from virus is acted upon from dicer to form siRNAs. siRNA forms a complex with RISC. RISC is now able to recognise RNA from target virus

27
Q

What are the differences between siRNA and miRNA?w

A

miRNAs are made from stem-loop RNAs with imperfect complementarity
siRNAs are made from dsRNAs with perfect complementarity

miRNAs are encoded from purposefully expressed endogenous genes
siRNAs are derived from exogenous or unwanted endogenous RNAs

miRNAs often show imperfect complementarity to target mRNAs
siRNAs often show perfect complementarity to target mRNAs

miRNAs are used to control gene expression
siRNAs are used as a defence against viral RNAs and some unwanted endogenous RNAs

28
Q

How are siRNAs used in the laboratory and in medicine?

A

Genes can be silenced using chemically synthesised siRNAs. Introduction of such an siRNA into cultured cells leads to degradation of >85% of the target mRNA

siRNAs are routinely used in the laboratory to silence expression of genes in cells growing in culture

siRNAs have huge promise in medicine

  • inhibit expression of viral genes
  • inhibit production of enzymes involved in cholesterol synthesis
29
Q

What is splicing?

A

Splicing is the removal of introns and joining of exons

only happens in eukaryotes as prokaryotes don’t have introns

30
Q

What are the cis acting sequences involved in splicing?

A

5’ GU (donor) and 3’ (acceptor) splice site sequences - these sequences are part of the intron and not the exon

Branchpoint - is an A residue that is followed usually by a run of T’s that is ~100 nucleotides before the end of the intron

31
Q

What are the trans-esterification reactions involved in splicing and what is the process of this?

A

Trans-esterification reactions refers to the breakage of phosphodiester linkages in pre-mRNA and new ones being formed.

The process:
- first reaction: -OH group of branchpoint is nucleophillic and attacks the phosphodiester bond between the first G of the intron and final nucleotide of the exon. A new bond is formed with the first G of the introns and the A of the branchpoint making a 3 way junction

  • second reaction: 3’ OH of exon not attacks the phosphoryl group at 3’ acceptor splice site. Joins the two exons and releases intron lariat
32
Q

What are the two types of splicing?

A
  • spliceosome dependant –> needs spliceosome

- spliceosome independent –> doesn’t need spliceosome

33
Q

Why are splice sites sequences at exon-intron junctions important?

A

Spliceosome is a huge machine which is composed of more than 200 proteins and 5 RNAs (snRNAs)

The splice sites interact with the spliceosome via its RNA components

34
Q

What are the 5 spliceosomal RNAs (snRNAs) and what do they interact with?

A

The 5 snRNAs are U1, U2, U4, U5 and U6 and associate with Sm proteins to form RNA-protein complexes (snRNP)

U1 –> partial complementarity to 5’ splice site
U2 –> partial complementarity to branchpoint/interacts with U6 snRNA
U4 –> binds to U6 snRNA to form U4/U6 snRNA
U5 –> to exon termini, positions exons during splicing
U6 –> later in spliceosome assembly interacts with U2 snRNA and 5’ splice site

35
Q

What is constitute and alternative splicing?

A

Constitutively spliced RNA: always identical mRNA sequences

Alternatively spliced RNA: different mRNA sequences

36
Q

What are the steps of spliceosome assembly?

A

U1 snRNA (component of snRNP) base pairs to 5’ splice site
U2 subsequently base pairs with branch site with A residue excluded
When U4/5/6 snRNP join spliceosome, U4 and U6 snRNA are heavily base paired
Unwinding of U4/U6 helix (and loss of U4) activates the spliceosome
It allows U6 to pair with U2
U1 snRNA departs (is unwound) and U6 also pairs with 5’ splice site
U5 interacts with both the end of first exon and start of second exon and holds them together after formation of lariat

37
Q

What groups of introns are spliceosome independent?

A

Group I and group II introns are spliceosome independent and so they self-splice as they have enzymatic activity. They are ribozymes

38
Q

What is translation?

A

Translation is the process by which genetic information contained within the order of nucleotides in the mRNA is interpreted to generate the linear sequence of amino acids in proteins

Translation can be thought to be gene expression regulation in the cytoplasm of eukaryotes

39
Q

What is the translation machinery primarily composed of?

A

tRNAs
Amynoacyl-tRNA synthetases (for charging tRNAs)
mRNAs
Ribosome

40
Q

What is the function of tRNA?

A

tRNA act as adaptors between the mRNA codons and the amino acid they are bound to

  • single stranded sections if loops can base pair with other RNA or molecules
  • amino acid is charged on acceptor arm
  • anticodon loop has 3 nucleotides that will be complementary to the nucleotide triplets on mRNA and will decide what amino acid to add
  • acceptor arm cannot bind amino acid by itself, it needs charging
41
Q

Why is charging of tRNA important?

A

The ribosome is unable to discriminate between correctly and incorrectly charged tRNAs

42
Q

What enzyme charges the tRNA molecules with the correct amino acid?

A

Amynoacyl-tRNA synthetase

Accurate charging of tRNA to the correct amino acid is completed by amynoacyl-tRNA synthetase
Most organisms have 20 diffenrent tRNA synthetases with each of the different amino acids being attached to the appropriate tRNA by a single dedicated tRNA synthetase

43
Q

How does tRNA synthetase charge tRNAs with appropriate amino acid?

A

tRNA synthetases recognise unique structural features of cognate tRNAs. A key determinant is the acceptor stem in tRNA which is specific for each amino acid

1) adentylation - adenosine monophosphate is added to the amino acid from ATP
2) tRNA charging - amino acid is joined to the tRNA with the release of AMP

44
Q

What is the composition of the eukaryotic ribosome?

A

Ribosome is composed of small subunit (40s) and large subunit (60s).

45
Q

What steps are involved in translation?

A

Initiation
Elongation
Termination

46
Q

Preparation of the ribosome

A

The ribosome is recruited to the mRNA to translate as the 40s subunit
40s binds with several eIFs (eIF1 + eIF1A + eIF3 that prevents re-association with large 60s subunit
Ternary complex: eIF2 + GTP + tRNA18

40s ribosomal subunit + eIF2 ternary complex = 43s complex
This complex is formed between the RNA that has bound methionine

47
Q

What role does the CAP have in recruiting the ribosome to mRNA?

A

The CAP-binding complex eIF4F is composed of 4 eIFs:

  • eIF4E –> binds to 5’ CAP
  • eIF4G –> interacts with eIF4E
  • eIF4A/ eIF4B –> interacts with eIF4E (4A has helicase activity, 4B activates 4A activity)

eIF4G interacts with eIF3 of 43s preinitiation complex and brings it to the mRNA

48
Q

How does preinitiation complex search for initiation codon?

A

Ribosome moves over mRNA with tRNA scanning sequence for start codon

  • dissociation of 48s complex from eIF4E to scan mRNA and look for AUG start codon
  • eIF4A activity melts mRNA secondary structure
  • context of AUG is critical to be recognised as imitator codon (Kozak consensus sequence)
49
Q

What is the Kozak consensus sequence?

A

Sequence that allows the preinitiation complex to recognise initiator codon

A/G -3 of A (of AUG), G +4 of A (of AUG)

50
Q

How does the large subunit (60s) join to form the 80s complex?

A

Initiation factors bound to small subunit are released due to the hydrolysis of the GTP into GDP
This allows large subunit to become bound

51
Q

What is the closed loop model of translation initiation?

A

The closed loops model proposes that 5’ and 3’ ends of mRNA are physically joined together
eIF4E joined to the 5’ CAP is also bound to eIF4G which can also bind to PABP which can bind the PolyA tail at the end of the mRNA chain

52
Q

What is the difference between eukaryotic and prokaryotic transcription and translation?

A

The process of transcription and translation happen simultaneously as there is no nucleus

53
Q

Gene expression: prokaryotes vs eukaryotes

A

Prokaryotes:

  • one type of RNA pol for all RNAs
  • genes grouped in operons
  • gene contain no introns
  • mRNA are usually polycistronic
  • mRNA is not modified
  • transcription and translation occur simultaneously

Eukaryotes:

  • three diffen types of RNA pol for different RNAs
  • no operons per se
  • genes usually with introns = alternative splicing
  • mRNAs are usually monocystronic
  • mRNA is midfield at 5’ end and 3’ end
54
Q

What does polycistronic mean?

A

Polycistronic mRNA means that it has multiple open reading frames

55
Q

How is the ribosome recruited in prokaryotes?

A

Ribosome is recruited internally

  • to the ribosome binding site (RBS) of Shine-Dalgarno sequence –> GGAGG
  • RBS is located 3-9 bases upstream of the start codon
  • RBS is complementary to a sequence at the 3’ end of the 16s rRNA in the small ribosomal subunit (also referred to as anti-Shine-Dalgarno)
56
Q

Are shine-dalgarno sequences always complementary to the anti-shine-dalgarno sequence?

A

No, shine-dalgarno sequences aren’t always complementary to anti shine-dalgarno sequences. It is an idealised sequence that indicates the most frequently found base in each position.

The closer the sequence to the ideal consensus sequence, the better the mRNA will recruit the ribosome.

57
Q

How is translation in prokaryotes regulated?

A

Translation in prokaryotes is regulated by riboswitches. There are specialised domains in mRNA which act as on-off elements (riboswitches)

58
Q

What are riboswitches?

A

Riboswitches function as sensors for signals as diverse as temperature, salt concentration, metal ions, amino acids and other small organic metabolites.

The key mechanism of action of function of riboswitches rely on changes in RNA secondary structure

Change in structure of RNA as a result of metabolite interaction means the SD can or can longer bind with anti-SD sequence regulating translation by turning on and off respectively

59
Q

What is the mechanism of the Rho-independent terminator?

A

Termination occurs when an RNA polymerase transcribed through an terminator and:

  • a stem loop forms in newly synthesised RNA - this causes RNA polymerase to pause
  • the weak AU base pairs between transcript and template unwind, so RNA molecule is release and complex dissociates

Molecular Biology Of The Gene (6 decks)

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