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Principles of Molecular Biology > RNA, RNA Processing and Techniques to Study RNA > Flashcards

RNA, RNA Processing and Techniques to Study RNA Flashcards

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1
Q

What are 3 differences between RNA and DNA?

A
  1. RNA has the ribose sugar, which has a 2’ OH
  2. Uracil instead of thymine
  3. Single strand instead of double strand
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2
Q

Why can’t RNA form a double helix?

A

The 2’ OH

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3
Q

How is RNA able to have many more different functions than DNA?

A

It’s structure. It isn’t limited to the double helix, so it can take on many different 3D structures and perform many more functions

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4
Q

What base pairs exist in RNA?

A

The Watson-Crick base pairs of CG and AU, but RNA can also have GU and GA because it isn’t limited to the double helix. RNA can also form base triplets with 3 bases hydrogen bonding to each other

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5
Q

What are 5 types of RNA found in cells?

A

mRNA, tRNA, rRNA, miRNA, snRNA

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6
Q

What is the function of mRNA?

A

Carries the transcribed DNA to the ribosome to be translated

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7
Q

What is the function of tRNA?

A

Base pairs with the codon on the mRNA and brings the correct amino acid to be added to a growing protein chain during translation

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8
Q

What is the function of rRNA?

A

Makes up a lot of the ribosome and is involved with catalyzing the formation of the peptide bonds between amino acids

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9
Q

What is the function of miRNA?

A

Gene down-regulation

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10
Q

What is the function of snRNA?

A

Is involved in splicing to modify transcribed mRNA

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11
Q

What is the RNA world hypothesis?

A

The hypothesis that in early life forms, RNA evolved before DNA did. RNA can both store information and carry out cell functions, and if it was self-replicating it could be the genetic material and cell machinery before DNA and proteins came about

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12
Q

What are ribozymes?

A

RNA with a catalytic function. Found in self-splicing introns, spliceosomes, and ribosomes

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13
Q

What is the 5’ cap made of?

A

7-methylguanosine cap - a modified guanine attached to the 5’ end of the mRNA transcript

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14
Q

What type of bond attaches the 5’ cap to the mRNA transcript?

A

5’-5’ linkage. The 5’ phosphate of the modified guanine is attached to the 5’ phosphate of the first nucleotide in the mRNA with a triphosphate bridge in between

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15
Q

What is the purpose of the 5’ cap?

A

Protects the mRNA from degradation by exonucleases and is bound by cap proteins

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16
Q

What enzyme adds the polyA tail to the mRNA transcript?

A

PolyA Polymerase (PAP)

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17
Q

What is the polyA tail?

A

Hundreds of adenine nucleotides are added to the 3’ end of the mRNA transcript once it has been cleaved off from RNA polymerase

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18
Q

What is the purpose of the polyA tail?

A

Acts as a buffer to stop the desired sequence on the mRNA transcript from being degraded by exonucleases as the exonucleases just eat the polyA tail instead of the sequence we want. It is also bound by polyA tail proteins

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19
Q

What is splicing?

A

Removal of internal mRNA sequences and re-ligation of the remaining fragments

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20
Q

What are the parts of the mRNA that are removed called?

A

Introns

21
Q

What are the parts of the mRNA that stay and are translated called?

A

Exons

22
Q

What experiment provided experimental evidence for the existence of introns?

A

RNA and DNA were mixed together and the solution was heated so the DNA was denatured. Then the solution was cooled to allow the DNA and RNA to reanneal, and we can get DNA to anneal to RNA. They visualized the shapes with electron microscopy. If there were no introns, the RNA would perfectly base pair to the DNA and would get a linear fragment. If there were introns, only the exons would bind to the DNA and the introns would loop out of the way, which is what they saw

23
Q

What are the consensus sequences at the 5’ splice site, the 3’ splice site, and the branch point?

A

5’ splice site: GU
3’ splice site: AG
Branch point: A

24
Q

Why are the splice sites present in the introns and not the exons?

A

If they were in the exons, it would limit the amino acids that could be near introns, which would limit the functionality of the protein

25
Q

Why doesn’t the splicing machinery cut at every GU and AG?

A

The surrounding nucleotides are important for context and lets the splicing machinery differentiate between the splice site and not the splice site

26
Q

What are the steps in mRNA splicing?

A
  1. The A at the branch point attacks the G at the 5’ splice site
  2. The 5’ G is cleaved off from the exon and ligated to the A at the branch point
  3. The 3’ end of the 5’ exon attacks the G of the 3’ splice site
  4. The 3’ G gets cleaved off from the neighbouring 3’ exon
  5. The exons are ligated together and the intron forms a lariat structure
27
Q

What is a spliceosome?

A

The cell machinery that preforms mRNA splicing, made up of snRNPs

28
Q

What is a snRNP?

A

5 snRNAs and a few proteins that form a complex, which then assemble to form the spliceosome

29
Q

What is the function of the snRNAs in the spliceosome?

A

They complementary base pair to the pre-mRNA and are ribozymes. They catalyze the transesterification reaction

30
Q

What is the function of the proteins in the spliceosome?

A

They help the snRNA by positioning the RNA subunits for catalysis and remodelling the spliceosome, but they don’t catalyze the reaction

31
Q

What are self-splicing RNAs? Where are they found?

A

Might be the evolutionary precursor to the spliceosome, they are sequences within the mRNA that catalyze the transesterification. They are found in mitochondria, chloroplasts, some fungi, plants, and algae

32
Q

What can happen if splicing gets messed up by a mutation?

A

An exon can get cut out with an intron, or an intron might not get cut out. It is very easy to mess up, 15% of disease-related point mutations are from splicing defects

33
Q

What is required for processed mRNA to leave the nucleus?

A
  1. The polyA tail and polyA binding proteins bound to it
  2. The 5’ cap and cap binding proteins bound to it
  3. Bound to spliceosome proteins at the exon-exon boundaries
34
Q

What is a probe?

A

A short sequence of nucleic acids that are labelled and complementary to the sequence of interest

35
Q

Why do we use probes to detect DNA and RNA?

A

It allows us to detect a sequence of interest in a complex mixture

36
Q

How does Northern blotting work?

A

RNA in a solution is separated out on agarose gel and transferred to a solid membrane. A labelled probe for the sequence of interest is added to the membrane and visualized

37
Q

What can a Northern blot tell us?

A

If a sequence of interest is present or absent, the size of the sequence, to an extent how much is there (not very quantitative, can’t tell minute differences), and any splicing variants

38
Q

What is a Southern blot? What can it tell us?

A

Same as a Northern blot but for DNA. Can tell us positional information on a gene (for genome mapping) and if a specific DNA sequence is present or absent

39
Q

How does RT-PCR work?

A

RNA in a solution is reverse transcribed to cDNA. Then primers are added to amplify the sequence of interest and PCR is done on the cDNA. The result is run out on a gel

40
Q

What type of primer is used for RT-PCR for the reverse transcriptase?

A

The polyT primer. It binds to the polyA tail and works for all eukaryotic mRNA

41
Q

What does RT-PCR tell us?

A

If an mRNA of interest is present in the solution

42
Q

How does qPCR work?

A

The amplification of a DNA sequence of interest is tracked with fluorescence. The fluorescence is detected after each cycle instead of at the end like usual PCR, and can use the tracked fluorescence to determine how much DNA of that sequence was initially there

43
Q

How does q-RT-PCR work?

A

qPCR coupled to reverse transcription. The mRNA is reverse transcribed into cDNA, and then the cDNA is amplified and tracked with fluorescence

44
Q

What do qPCR and q-RT-PCR tell us?

A

They are both very quantitative and tell us how much of a sequence of interest was originally present in the sample

45
Q

How does a microarray work?

A

All mRNA in an organism undergoes RT-PCR with fluorescent nucleotides to create fluorescent cDNA. The cDNA is then hybridized to the DNA on the glass and will fluoresce when it binds to the complementary DNA on the microarray

46
Q

What do we typically use a microarray for?

A

To compare gene expression of all genes between two samples, usually earlier and later stages in development or drug treatment vs no drug treatment, diseased vs normal tissue, etc

47
Q

How are each of the samples labelled and interpreted on a microarray?

A

One sample is labelled green, the other is labelled red. On the microarray, if the green sample is the only one expressed the spot on the microarray will be green. If the red sample is the only one expressed the spot on the microarray will be red. If they are both expressing the spot on the microarray will be yellow

48
Q

How does in situ hybridization work?

A

The probe is added to the intact tissue or organism and is visualized

49
Q

What does in situ hybridization tell us?

A

Where the mRNA is being expressed

Principles of Molecular Biology (17 decks)

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