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BCHM218 > Module 4 Sections 4-7 > Flashcards

Module 4 Sections 4-7 Flashcards

Translation Initiation, Elongation, and Termination in Prokaryotes and Eukaryotes, Regulation of Gene Expression, Post-transcriptional Gene Silencing (PTGS)

1
Q

tRNA

A

recognizes specific codons within the mRNA sequences and carries the required amino acid to the growing polypeptide and transfers the RNA to protein

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2
Q

structure of tRNA

A
  • non-coding
  • small
  • single stranded RNA, 73-93 nucleotide residues long
  • folds back on itself to produce a secondary structure - a result of intramolecular base pairing within the single RNA strand
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3
Q

tRNA amino acid arm

A

Has a trinucleotide sequence CCA at the 3’ terminus

The A residue is the nucleotide to which the amino acids attach

Each tRNA will carry a specific amino acid, making it amino acylated

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4
Q

amino acylated

A

has an amino acid bound to it

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5
Q

tRNA anticodon arm

A

At the opposite end, there is the anticodon

3-nucleotide sequence that base pairs with the complementary mRNA

The base pairing between the anticodon in the tRNA and the codon in the mRNA is complementary

Ex: codon for methionine = 5’-AUG base pairs with the tRNA Met anticodon 5’-CAU (3’-UAC)

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6
Q

aminoacyl-tRNA synthetases

A

enzymes that are attached to the amino acids to particular tRNAs to provide the certain specificity for the correct amino acid

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7
Q

what energy does aminoacyl-tRNA synthetases use

A

ATP

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8
Q

why are aminoacyl-tRNA synthetases needed

A

anticodon is positioned 70 A residues away from the 3’ terminus of the amino acid arm of the tRNA, it is too far to specify the correct amino acid for a given tRNA

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9
Q

wobble base pairing

A

some tRNAs can recognize more than 1 codon, so there are less number of tRNAs needed in a cell (less than 61, since there are 61 possible codons)

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10
Q

adenosine deaminase acting on RNA (ADAR)

A

converts adenosine to inosine

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11
Q

inosine

A

converted from andenosine from ADAR, can form wobble base pairs with A, C, or U in the 3rd position of the codon

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12
Q

how many tRNAs are required to translate all 61 codons

A

32 = 31 for the amino acids and 1 for initiation

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13
Q

the wobble hypothesis

A

First 2 bases of an mRNA codon always form Watson-Crick base pairs with the corresponding bases of the tRNA anticodon

First base of the anticodon (5’-3’) pairs with the 3rd base of the codon and determines the number of codons recognized by the tRNA:

C or A = tRNA recognizes one codon
U or G = 2 codons

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14
Q

where does the interaction between tRNA and mRNA occur

A

the ribosome

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15
Q

the ribosome

A
  • an RNA enzyme
  • macromolecular complex of rRNAs and r-proteins
  • function as the protein factories of the cell
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16
Q

where is the ribosome found

A

free in the cytoplasm or bound to the endoplasmic reticulum

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17
Q

what is the ribosome composed of

A

RNA and protein

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18
Q

what is the ribosomal RNA responsible for

A

the functional activity of the ribosome

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19
Q

peptidyl transferase center

A

in the 60S subunit (larger), catalyzes peptide bond formation between adjacent amino acids

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20
Q

decoding center - in the 40S (smaller) subunit

A

amino acylated tRNAs read the genetic code by base pairing with each triple codon in the mRNA

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21
Q

relationship between amino acids, tRNAs, and the ribosome

A

All involved in translation

Amino acids are the building blocks of proteins

tRNAs function as adaptor molecules

Carry specific amino acids to the ribosome where they are added to the growing polypeptide chain

Aminoacyl-tRNA synthetases provide the specificity of tRNA for a specific amino acid

The ribosome is the protein factory within the cell

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22
Q

ribosomal binding sites

A
  1. A site
    - Aminoacyl-tRNA binding (charged tRNAs)
  2. P site
    - peptidyl-tRNA binding (tRNAs that contains the growing polypeptide chain)
  3. E site
    - exit site, occupied by the tRNA molecule released after the growing polypeptide chain is transferred to the aminoacyl-tRNA in the P site
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23
Q

steps of translation

A
  1. Initiator tRNA is charged with methionine
    - Both bacteria and eukaryotes have 2 forms of tRNA for methionine
    - One for initiation of translation and one for the insertion of methionine into a growing peptide chain
  2. Translation initiates with the assembly of mRNA and amino acylated tRNA on the small ribosomal subunit, followed by joining with the large subunit to form an active ribosome
  3. Polypeptide elongation
    - Occurs in successive cycles of aminoacyl-tRNA binding and peptide bond formation in the order directed by the genetic code in the mRNA
  4. Translation termination
    - Occurs when the ribosome encounters a stop codon in the mRNA
    - Releases the mRNA and dissociates the ribosome into its 2 subunits
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24
Q

initiation of translation

A
  1. alignment of mRNA on the small ribosomal unit
    - IF-3 associates with the small subunit to prevent the premature assembly of the ribosome
  2. aossication of a charged initiator tRNA with the AUG start codon in the P site
    - This tRNA is guided to the ribosome by IF-2
    - IF-1 blocks the A site to ensure the correct alignment of the tRNA with the AUG start codon
  3. Recruitment of the large ribosomal subunit to form a complete initiation complex
    -IFs dissociate from the complex (consumes GTP)
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25
Q

GTP

A

energy currency of translation

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26
Q

IF-3

A

associates with the small subunit to prevent the premature assembly of the ribosome

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27
Q

IF-2

A

guides the charged initiator tRNA with the AUG start codon in the P site

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28
Q

IF-1

A

blocks the A site to ensure the correct alignment of the tRNA with the AUG start codon

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28
Q

shine-dalgarno sequence

A

guides the initiating 5’-AUG to its correct position

Signal of 4-9 purine residues, situated 8-13 nucleotides on the 5’ side of the start codon

The sequence base pairs with a complementary pyrimidine-rich sequence near the 3’ end of the 16S rRNA of the small ribosomal subunit which positions the 5’-AUG sequence of the mRNA in the precise location on the 30S subunit, where it is required for translation initiation

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29
Q

tRNA (fMet)

A

The amino acid incorporated in response to the 5’-AUG initiation codon

Formed by methionine attaching to tRNA(fMet) by the Met-tRNA synthetase, and also a transformylase enzyme transferring a formyl group to the amino group of the methionyl part of the tRNA

Addition of the formyl group = fMet residue cannot be added internally (the N group is blocked)

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30
Q

tRNA (Met)

A

Used to bring in methionine residue when there is an AUG codon within the mRNA transcription (not at the 5’ initiation position)

The absence of the N-formyl group enables it to insert a methionine residue at internal positions within the growing polypeptide chain

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31
Q

tRNA fMet vs tRNA Met

A

tRNA fMet is charged with N-formylmethionine and this amino acid is incorporated in response to the 5-AUG initiation codon

tRNA Met is charged with the amino acid methionine and inserts the Met residue internally within the polypeptide chain

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32
Q

polycistronic mRNA

A

a contiguous mRNA with more than 2 genes that can be translated into proteins

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33
Q

are bacterial and eukaryotic mRNAs polycistronic or monocistronic

A

bacterial = poly
eukaryotic = mono

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34
Q

monocistronic

A

encodes for a single protein

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35
Q

bacterial genes can be both

A

overlapping or non-overlapping

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36
Q

non-overlapping bacterial genes

A

The open reading frame for each gene is distinct from one another and they will have separate Shine-Dalgarno sequences

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37
Q

overlapping bacterial genes

A

Despite lacking a Shine-Dalgarno sequence for each internal start site, the internal open reading frames can be translated efficiently because of overlapping start and stop codons, usually 5’-AUGA

Ribosomes terminating translation of the upstream message can initiate the downstream message simply by shifting their reading frame

Overlapping genes
Shine-Dalgarno sequence Protein-coding region I
Protein-coding region 2

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38
Q

kozak sequences

A

sequence around the start codon in eukaryotic mRNA that guides translation - has a purine nucleotide 3 residues before, and a G residue immediately after the start codon

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39
Q

where are kozak sequences

A

surrounds the initiation site

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40
Q

characteristics of kozak sequences

A

enhance translation through contact with the eukaryotic initatior tRNA through its anticodon arm

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41
Q

polysome

A

a single mRNA transcript bound by multiple ribosomes

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42
Q

eIF4F

A

subunits of the eukaryotic initiation factor

interacts with either the 5’ cap or poly(A) binding protein (PABP) which is associated with the 3’ poly(A) tail of the mRNA molecule

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43
Q

PABP

A

poly(A) binding protein

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44
Q

functions of eIF4F

A

Ensuring that mRNA processing is complete prior to translation

Promoting translational efficiency

Enabling the sophisticated translational regulation of gene expression

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45
Q

elongation general overview

A

The nascent polypeptide is lengthened by the covalent attachment of successive amino acid units

Each unit is carried to the ribosome and correctly positioned by its tRNA which base pairs to its corresponding codon in the mRNA

Energy currency – GTP (guanosine triphosphate) and not ATP

Dipeptidyl-tRNA = a tRNA carrying a growing peptide chain of 2 peptides

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46
Q

steps of elongation

A
  1. binding of an aminoacyl-tRNA in the A site
  2. peptide bond formation between the polypeptide in the P site and the amino acid in the A site, transferring the growing polypeptide chain to the tRNA in the A site
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47
Q

EF-Tu-GTP

A

delivers a charged tRNA to the A site of an active ribosome at the decoding center of the ribosomal complex

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48
Q

what energy does EF-Tu-GTP use

A

the energy is provided by the hydrolysis of EF-Tu-GTP to EF-Tu-GDP + Pi

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49
Q

accommodation

A

occurs when the correct codon base pairs with an anticodon and the ribosome changes configuration

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50
Q

result of an incorrect aminoacyl-tRNA in the A site

A

gets dissociated

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51
Q

result of accommodation

A

release of EF-Tu-GDP

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52
Q

EF-Tu-GDP recycling process

A

EF-Tu-GDP is recycled to EF-Tu-GTP through the actions of the EF-Ts: the guanine nucleotide exchange factor for EF-Tu

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53
Q

the action of the EF-Ts

A

guanine nucleotide exchange factor for EF-Tu

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54
Q

what happens after EF-Tu-GTP deliveres a charged tRNA to the A site

A

2 adenosine residues (A1492 and A1493) “flip out” in response to correct codon-anticodon base pairing

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55
Q

steps of elongation detailed

A
  1. tRNAs are delivered to the A-site by GTP-bound EF-Tu
  2. A1492 and A1493 of the ribosomal A-site “flip out” in response to correct codon-anticodon base pairing charged
  3. EF-Tu-GTP is hydrolyzed to EF-Tu-GDP + Pi
  4. tRNA rotates into position - accommodation
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56
Q

the peptyidyl transferase reaction

A

a peptide bond is formed between the 2 amino acids bound by their tRNAs to the A and P sites on the ribosome

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57
Q

steps of the peptidyl transferase reaction

A
  1. Formation of the first peptide bond occurs through the transfer of the initiating N-formyl methionyl group from its tRNA in the P site to the amino group of the second amino acid on its tRNA in the A site
  2. A nucleophilic attack of the alpha-amino group of the A-site aminoacyl-tRNA on the carbonyl carbon of the ester bond linking the fMet (or the growing peptide chain) to the P-site tRNA forms the peptide bond
  3. The growing chain is transferred to the tRNA in the A site
    - As the ribosome shifts along the mRNA, the uncharged tRNA now moves to the E site and the peptidyl-tRNA moves to the P site
    - This frees the A site to bind the next tRNA
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58
Q

antibiotics

A

produced by bacteria/other microorganisms to inhibit protein synthesis in other bacteria

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59
Q

classes of bacterial antibiotics

A
  1. aminoglycosides
  2. puromycin (aminonucleosides)
60
Q

aminoglycosides’ effect in translation

A

reduce translational accuracy - Binds specifically to the decoding center of the small ribosomal subunit and causes inappropriate flipping A1492/1493 even when an incorrect tRNA is positioned in the A site

Kill bacteria through errors in translational fidelity by causing misfolded proteins and eventually cell death

61
Q

aminoglycosides are composed of

A

gentamicin, streptomycin and paromomycine

62
Q

puromycin’s effect on translation

A

mimics aminoacyl tRNA structure and prematurely stops protein synthesis

63
Q

puromycin structure

A

similar to the 3’ end of an aminoacyl-tRNA so it can bind to the reibosomal A site and participate in peptide bond formation

Since it only resembles the 3’ end of the tRNA, it cannot engage in translocation and dissociates from the ribosome shortly after it is linked the C-terminus of the peptide

64
Q

aminonucleosides vs aminogylcosides

A

aminonucleosides: binds to the ribosomal A site and participates in peptide bond formation, but does not engage in translocation, stopping protein synthesis

aminoglycosides: binds the small subunit (like EF-Tu) and causes inappropriate flipping of A1492/1493

65
Q

what is required for translation termination

A

release factors (RF)

66
Q

release factors for prokaryotes

A

RF-1 and RF-2

67
Q

release factors for eukaryotes

A

eRF1

68
Q

RF-1 and RF-2 jobs

A

Recognize termination codons and bind the A-site of the ribosome in much the same way as tRNAs

RF-1 recognizes stop codons UAG and UAA

Rf-2 recognizes UGA and UAA

Either one binds at the stop codon and induces peptidyl transferase to transfer the growing polypeptide to a water molecule rather than to another amino acid

69
Q

the lac operon

A

transcriptional regulation

70
Q

why we use bacteria to study gene expression

A

Haploid, so the effects of gene mutations are unmasked

Easy to see the impact of a single mutated gene on bacterial function

Need to initially understand gene regulation in simpler organisms to create networks of greater magnitude in other organisms

71
Q

operon

A

unit of genetic expression consisting of 1 or more cotranscribed genes and the operator and promoter sequences that regulate their transcription

72
Q

2 defining characteristics of bacterial operons

A
  1. A set of genes (A, B, and C) transcribed as a single mRNA
  2. Adjacent regulatory regions that coordinately control the expression of the operon genes
    - Can contain the same promoter, activator, and repressor regions as genes
73
Q

the lac operon composition

A

Polycistronic mRNA, containing 2 regulatory regions (lacl, lacO) and 3 genes that are referred to as the lac genes (lacZ, lacY, and lacA)

74
Q

when is lac operon turned on/off

A

When lactose is available, the lac operon is turned on by the regulatory regions, and the 3 lac genes are expressed. When unavailable, transcription from the lac operon is greatly reduced

75
Q

lacI

A

Encodes the lac repressor protein, which interacts with lacO to regulate transcription

Repressor is transribed separately from the rest of the operon (has a separate promoter) and is always on

76
Q

lacO

A

Lac operator

Does not code for a gene product, but instead interacts with lacI to regulate transcription

77
Q

lacZ

A

Codes for the protein B-galactosidase, which catalyzes cleavage of lactose into its components, glucose and galactose that can be further metabolized to generate ATP

78
Q

isomerization

A

lactose to allolactose

79
Q

what causes isomerization

A

B-galactosidase

80
Q

lacY

A

Codes for the galactoside permease protein, which inserts into the bacterial plasma membrane and imports lactose into the cell

81
Q

lacA

A

Codes for the protein thiogalactoside transacetylase, which modifies toxic galactosides that are imported along with lactose, facilitating their removal from the cell

82
Q

thigalactoside transacetylase

A

protein that modifies toxic galactosides that are imported along with lactose, facilitating their removal from the cell

83
Q

bacterial conjugation

A

Phenomenon by which DNA is transferred from a donor to a recipient cell. The transfer is mediated by a genetic element, separate from the bacterial chromosome, known as the F plasmid)

84
Q

F plasmid

A

mediates the transfer of DNA from a donor to a recipient cell

85
Q

merodiploid model

A

partially diploid bacterium which has its own chromosome complement and a chromosome fragment introduced by a process such as conjugation

86
Q

experiment 1 of lac operon

A

using lac operon mutant e.coli to understand the mechanisms of gene regulation

87
Q

experiment 1 of lac operon conclusions

A

Hypothesized that the lacl locus produced a diffusible product that could act on any DNA molecule, not just from the DNA which it was generated

88
Q

experiment 2 of lac operon

A

examining the role of the lac operator

89
Q

experiment 2 of lac operon conclusions

A

The lacO did not produce a diffusible substance

The lac operon DNA from the lacO mutant could not be correctly regulated even in the presence of a wild-type copy of lacO

LacI functioned like a transmitter

If knocked out, it could be replaced by a second transmitter

LacO functioned like a receiver

If a message could not be received, the action (transcription of the lac genes) could not be controlled

90
Q

experiment 3 of the lac operon

A

examining the role of the lac operator part 2

91
Q

experiment 3 of the lac operon conclusions

A

Confirmed that the lacI gene encodes a diffusible molecule (acts in trans) that represses lac gene expression, whereas lacO controls only the expression of lac operon genes to which it is connected (acts in cis)

92
Q

lacI gene acting in trans

A

encodes a diffusible molecule

93
Q

lacI gene acting in cis

A

controls only the expression of lac operon genes to which it is connected

94
Q

negative regulation

A

binding of a repressor protein that prevents or decreases expression

ex: the binding of the lac repressor to the lac operator

95
Q

the lac repressor purpose

A

required in order to block gene transcription

96
Q

when does the lac repressor occur

A

in the absence of lactose, which means that the bacteria is not wasting energy to produce the protein products of the lac operon required for lactose metabolism when there is no lactose present to metabolize

97
Q

lac repressor structure

A

Homotetrameric DNA binding protein

2 dimers bound together at the end opposite to the DNA binding region

Binding region contains a helix-turn-helix motif at the N-terminus, allowing it to bind to the major groove of DNA

98
Q

homotetrameric

A

4 identical subunits in a protein

99
Q

lac repressor binding site/operator regions

A
  1. 3’ of promoter region
  2. in lacZ gene
  3. 5’ of promoter
100
Q

inverted repeat

A

a single stranded sequence of nucleotides followed downstream by its reverse complement

101
Q

leaky expression

A

repression via the lac repressor is not 100% effective

102
Q

how can the lac operon be activated if there is no lactose and repression occurs?

A
  1. leaky expression
  2. Binding of the repressor reduces the rate of transcription initiation by a factor of 1000, but even in the repressed state, each cell has a few molecules of B-galactosidase and galactoside permease, presumably synthesized on the rare occasions when the repressor transiently dissociates from the operators
  3. When cells are provided with lactose, the few existing molecules of galactoside permease enable lactose from the medium to enter the cell
103
Q

allolactose

A

function as effectors under circumstances where activation of operon is needed

104
Q

role of effectors in negative regulation

A

effector = a molecular signal that regulates the binding of a repressor to DNA (like allolactose)

105
Q

conformation change on repressor results in

A

A small molecule or protein binds the repressor and causes a conformational change that results in an increase or decrease in transcription

106
Q

activation in negative regulation

A

The effector binds to the repressor and induces a conformational change that results in dissociation of the repressor from its binding site on the DNA, allowing transcription to proceed

107
Q

inactivation in negative regulation

A

the interaction of an inactivating repressor and an effector molecule causes the repressor to bind to DNA, shutting down transcription

108
Q

allolactose

A

an allosteric effector

109
Q

allolactose purpose

A

Induces the lac operon by binding to a specific site on the lac repressor, causing a conformational change that results in dissociation of the repressor from the operator

110
Q

when is there positive regulation of the lac operon

A

In the presence of glucose, the operon is blocked by catabolite repression, a form of positive regulation

111
Q

catabolite repression

A

the inhibition of the expression of genes required for the metabolism of, in the case of the lac operon, other sugars in the presence of glucose

112
Q

cAMP receptor protein (CRP)

A

activator protein that regulates positive regulation of the lac operon

113
Q

why are activators needed

A

Transcription is weak without an activator interacting with RNA polymerase to enhance DNA binding and transcriptional initiation

114
Q

is CRP a homodimer

A

yes, each subunit binds 1 molecule of cAMP, which is required for binding of CRP to the activator binding site of the lac operon DNA

115
Q

how is adenosine nucleotide produced

A

by an enzyme called adenylyl cyclase (ATP —> cAMP + pyrophosphate)

116
Q

the role of effectors in positive regulation

A

Effectors in positive regulation bind an activator, rather than a repressor, to cause a conformational change that results in an increase or decrease in transcription

117
Q

activation in positive regulation

A

cAMP is required for binding of the activator (CRP) to the activator binding region of DNA, increasing expression

118
Q

inactivation in positive regulation

A

binding of the effector reduces the affinity of the activator for DNA, and inhibits expression

119
Q

cAMP is directly regulated by

A

glucose

120
Q

in the presence/absence of glucose:

A

In the presence of glucose, cAMP production is decreased & cAMP is exported from the cell

In the absence of glucose, cAMP production is increased and cAMP is retained in the cell

121
Q

glucose present, lactose present

A

lactose availability causes repressor to dissociate

low cAMP levels prevent CRP binding, meaning RNA polymerase may only occasionally initiate transcription

122
Q

glucose present, lactose absent

A

cAMP is low and the cAMP-CRP does not bind the operon. the repressor binds the operator, blocking RNA polymerase and preventing transcription of the lac genes

123
Q

glucose absent, lactose present

A

when glucose is absent and lactose is present, the activator binds a different small molecule, which causes it to bind DNA and recruit RNA polymerase for high-level gene expression

124
Q

housekeeping genes in eukaryotes

A

reqjired at all times, expressed at a constant level

125
Q

regulated gene expression in eukaryotes

A

Undergo activation and repression to changes in environmental conditions

Require the assistance of transcription factors, and the proteins that alter the affinity of the RNA polymerase for the promoter

Enhance gene expression = activators

Reduce expression = repressors

Regulators act by binding to specific DNA sequences known as regulatory sites, and either facilitating or inhibiting transcription

126
Q

positive regulation

A

binding of a common transcriptional activator

127
Q

how is positive regulation produced

A

from scratch by expression of its gene or an existing activator protein may become active for DNA binding through interaction with another protein or a small effector molecule

128
Q

regulation by destruction of repressors

A

Removal of a common repressor bound to DNA sites, either by an allosteric change induced by binding of a small effector molecule, or by proteolytic digestion of the repressor

129
Q

allosteric change

A

change in shape/conformation of binding site

130
Q

DNA looping

A

Activator binds to a DNA binding region distant from the promoter, and the DNA is able to fold over so that it can make contact with the transcriptional machinery

131
Q

coactivators and corepressors

A

Proteins that can make protein-protein interactions to influence transcription - don’t bind to DNA directly, just other proteins

132
Q

coactivators

A

Act as a bridge between an activator and the RNA polymerase to activate transcription

133
Q

corepressors

A

Act as a block to inhibit binding of the activator to the RNA polymerase and prevent transcription

134
Q

bacteria vs eukaryotes

A

have methods to regulate groups of genes simultaneously: both

often have several activator and repressor binding sites per gene: eukaryotes

have polycistronic genes: bacteria

use distant regulatory sites that act through DNA loops: eukaryotes

are constantly changing the activation and repression states of geenes in response to current needs and evnironmental conditions: both

135
Q

DNA binding proteins often recognize the major or minor groove

A

major groove

136
Q

DNA binding motifs

A

can form heterodimers composed of 2 different members of a family of similarly structured proteins, creating a larger number of functional transcription factors

137
Q

what do DNA binding motifs / heterodimers provide

A

This provides combinatorial control of gene expression (The use of specific combinations of limited number of regulatory proteins to exert fine control over gene expression)

138
Q

post-transcriptional gene silencing (PTGS)

A

Genes are still transcribed but the resulting mRNAs are degraded before they can be translated into proteins

139
Q

RNA interference (RNAi) hypothesis

A

base pairing between the antisense oligonucleotide and the target mRNA would prevent recognition by the translation machinery or lead to degradation of the hybrid complex, or both

140
Q

RNA interference (RNAi) conclusion

A

Careful analysis in the roundworms revealed that protein production was intercepted in the worms after the injection of double stranded RNA

when injected with sense or antisense strand alone = normal worms

when injected with dsDNA = worms had a twitching phenotype

141
Q

miRNAs processing in the cell steps

A
  1. miRNAS are first transcribed as primary miRNA transcripts (pri-mRNAs) with 1 or more sets of internally complementary sequences that can fold to form hairpin-like structures
  2. The pri-miRNAs are cleaved by nuclear endonuclease Drosha to produce shortened hairpins (60-70 nucleotides) with a 5’ phosphate and a 2-nucleotide 3’ overhang
    - Now known as precursor miRNAs (pre-miRNAs)
  3. Pre-miRNAs then bind to export receptor proteins and are transported from the nucleus to the cytoplasm for further processing
142
Q

what is required for gene silencing

A

siRNA and miRNA

143
Q

silencing a gene steps

A
  1. In the cytoplasm, pre-miRNAs are cleaved by the Dicer ribonuclease to generate a 20-22 nucleotide ds miRNA. They are then incorporated into a complex called the RNA-induced silencing complex (RISC) which includes the Argonaute (AGO protein)
  2. After cleave, the miRNA is unwound and the unneeded passenger strand is discarded
  3. The strand complementary to the target is then delivered to a particular mRNA by the AGO protein
  4. Once the mRNA has been targeted, the degree of miRNA-mRNA base pairing leads to either Argonaute-mediated degradation (high complementarity) or translational repression (lower complementarity), followed by degradation
144
Q

what are pre-miRNAs cleaved by and what do they generate

A

cleaved by the Dicer ribonuclease to generate a 20-22 nucleotide ds miRNA

145
Q

miRNA in animals vs plants

A

animals = miRNAs are complementary to region in the 3’UTR in the mRNA
plants = in the coding region

146
Q

how to test the specificity of a miRNA

A
  1. Use a report plasmid to subclone the 3’UTR of the gene of interest downstream of a reporter gene
  2. Add the miRNA you want to test
  3. Monitor resulting reporter gene expression
  4. Loss of expression of your reporter gene = miRNA is binding
147
Q

lncRNA and circRNA functions

A
  1. compete as targets for miRNA binding
  2. non-coding RNAs that contain miRNA binding regions - when miRNAs bind to these RNAs instead of their target mRNA, there is a decrease in mRNA repression and increased translation

BCHM218 (4 decks)

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