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MOLECULAR BIOLOGY II > WEEK 2 (Ribozymes & RNA interference) > Flashcards

WEEK 2 (Ribozymes & RNA interference) Flashcards

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1
Q

How are macromolecules constructed?

A

By covalently linking small organic monomers/subunits into long chains or polymers

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2
Q

What is classed as evidence of an RNA world?

A

RNA catalysts/RIbozymes catalyse several fundamental reactions in modern cells

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3
Q

Describe the evidence that RNA arose before DNA in evolution

A
  • RIBOSE is readily formed from FORMALDEHYDE whereas DEOXYRIBOSE is harder to make and in present day cells is produced from ribose in a reaction catalysed by a protein enzyme -> Suggests that ribose PREDATES deoxyribose in cells
  • Sugar-phosphate backbone makes chains of DNA chemically more stable than chains of RNA -> greater lengths of DNA can be maintained without breakage
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4
Q

What are the properties of rDNA?

A
  • Clustered in one or a few regions of the genome
  • Human genome has five rDNA clusters each on a different chromosome
  • In a non-dividing interphase cell, the clusters of rDNA are gathered together as part of one or more irregularly shaped nuclear structures called NUCLEOLI that function in producing ribosomes
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5
Q

How many distinct ribosomal RNAs do eukaryotic ribosomes have?

A

Eukaryotic ribosomes have four distinct ribosomal RNAs, 3 in the large subunit and one in the small subunit

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6
Q

Where can the different ribosomal RNAs be found?

A
  • LARGE SUBUNIT = 28S & 5.8S & 5S
  • SMALL SUBUNIT = 18S
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7
Q

What is the difference between the ribosomal RNAs in the large subunit and in the small subunit?

A
  • 28S, 5.8S & 5S are carved by various nucleases from a single primary transcript called pre-rRNA which is synthesised by RNA Polymerase I which transcribes the pre-rRNA
  • 5S rRNA is synthesised from a separate RNA precursor outside the nucleolus by a different enzyme called RNA Polymerase III
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8
Q

Name the two post-transcription changes in pre-rRNA that differentiate it from other RNA transcripts

A
  • Large number of METHYLATED NUCLEOTIDES
  • Large number of PSEUDOURIDINE RESIDUES
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9
Q

What modifications happen to pre-rRNA?

A
  • ISOMERISATION of URIDINE to PSEUDOURIDINE
  • METHYLATION of RIBOSE at the 2’ site of the sugar
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10
Q

How is Uridine converted to Pseudouridine?

A

The N1 C1 bond is cleaved and the uracil ring is rotated through 120° which brings C5 of the ring into place to form the new bond with C1 of ribose

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11
Q

What catalyses pre-rNA’s post-transcriptional modifications?

A

A protein component of the snoRNPs (small nucleolar ribonucleoproteins) called DYSKERIN

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12
Q

What are Small nucleolar ribonucleoproteins (snoRNPs)?

A

Small nucleolar RNAs (snoRNAs) that are packaged with particular proteins and are used to process pre-rRNA

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13
Q

Describe the role of snoRNAs in the processing of pre-rRNA

A

1) snoRNPs begin to associate with the rRNA precursor before it is fully transcribed
2) The first RNP particle to attach to a pre-rRNA transcript contains the U3 snoRNA and more than two dozen different proteins
3) This catalyses the removal of the 5’ end of the transcript

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14
Q

Primary transcript for the rRNAs

A
  • 45S molecule
  • 13000 bases
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15
Q

What are the properties of a 5S rRNA?

A
  • About 120 nucleotides long
  • Present as part of the large ribosomal subunit of both prokaryotes and eukaryotes
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16
Q

Describe the Synthesis & Processing of the 5S rRNA

A

1) In eukaryotes, the 5S rRNA molecules are encoded by a large number of identical genes that are separate from the other rRNA genes and are located outside the nucleolus
2) Following synthesis, it is transported to the nucleolus to join the other components involved in the assembly of ribosomal subunits
3) Transcribed by RNA Polymerase III

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17
Q

What makes RNA Polymerase III unusual among the three polymerases?

A

It can bind to a promoter site located within the transcribed portion of the target gene

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18
Q

What are the properties of the tRNA found in plant and animal cells?

A
  • 50 different species
  • Each species is encoded by a repeated DNA sequence (Degree of repetition varies with the organism)
  • tRNAs synthesised from genes found in small clusters scattered around the genome
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19
Q

What does a single cluster of genes encoding tRNAs contain?

A

Multiple copies of different tRNA genes & the DNA sequence encoding given tRNA is found in more than one cluster

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20
Q

What does the DNA within a cluster (tDNA) contain?

A

Nontranscribed spacer sequences with the tRNA coding sequences situated at irregular intervals in a tandemly repeated arrangement

21
Q

What makes the tRNAs similar to 5S rRNA?

A

Both are transcribed by RNA Polymerase III and the promoter sequence lie within the coding section of the gene rather than being located at its 5’ flank

22
Q

What happens to the primary transcript of tRNA once it is transcribed

A
  • Larger than the final product so pieces on both 5’ and 3’ sides of the precursors tRNA are trimmed away
  • Numerous bases are also modified
  • ribonuclease P (an endonuclease) is involved in pre-tRNA processing
23
Q

What are the properties of ribonuclease P?

A
  • Involved in pre-tRNA processing
  • Present in both bacterial and eukaryotic cells
  • Consists of RNA and protein subunits
  • RNA subunit of ribonuclease P that catalyses cleavage of pre-tRNA substrate
24
Q

What are the properties of Small RNA?

A
  • Complexed with proteins to form RIBONUCLEOPROTEINS
  • Distributed in the nucleus, cytoplasm or in both
  • Range in size from 20 to 300 nucleotides
  • Present in 100,000 to 1,00,000 copies per cell
25
Q

What are the properties of Small Nuclear RNAs (snRNAs)?

A
  • Involved in the process of splicing
  • Forms complexes with proteins to form RIBONUCLEOPROTEIN particles called snRNPs
26
Q

What are the properties of Eukaryotic ribosomes?

A
  • Large complex of four rRNAs and more than 80 small proteins
  • Prokaryotic ribosomes are very similar
  • Both Eukaryotic and Prokaryotic ribosomes are formed from a LARGE and SMALL subunit which only come together after the small subunit has bound an mRNA
  • Ribosomal proteins greatly outnumber rRNAs
  • RNAs account for most of the mass of the ribosome and give it its overall SHAPE and STRUCTURE
27
Q

Describe the structure of ribosomes

A
  • Each ribosome has a binding site for mRNA and three binding sites for tRNA
  • rRNAs are bound in the A site (AminoacyltRNA), P site (Peptidyl-tRNA) and E site (Exit)
  • During protein synthesis, only two sites are occupied at any one time
28
Q

What are the tRNA binding sites on the ribosome primarily formed by?

A

rRNAs

29
Q

What is the catalytic site for peptide bond formation formed by?

A

23S rRNA of the large subunit

30
Q

What is the catalytic site in the rRNA called?

A

Peptidyl Transferase

31
Q

Describe Peptide Transferase

A

The catalytic site in rRNA which is a highly structured pocket that precisely orients the two reactants (elongating polypeptide & charged tRNA) greatly increasing the probability of a productive reaction

32
Q

What are Ribozymes?

A

RNA molecules that possess catalytic activity

33
Q

Do RNAs have the same structural and functional diversity as protein enzymes?

A

NO they are built from only four subunits

34
Q

Which activities can in vitro selected RNAs catalyse?

A
  • DNA ligation
  • RNA Polymerisation
  • RNA Phosphorylation
  • RNA aminoacylation
  • RNA alkylation
  • C-C bond rotation (isomerisation)
35
Q

Which activity can ribosomal RNA catalyse?

A

Peptide bond formation in protein synthesis

36
Q

Which activity can Self-splicing RNAs and small nuclear RNAs catalyse?

A

RNA splicing

37
Q

Which reactions have been found to be catalysed by naturally occurring RNAs?

A
  • The cleavage and ligation of phosphodiester bonds required for RNA splicing
  • The formation of peptide bonds during protein synthesis
38
Q

What are the four control stages during the DNA-mRNA-protein pathway?

A

1) Transcriptional control
2) RNA processing control
3) RNA transport control
4) Translation control

39
Q

What is RNA interference (RNAi)?

A

A biological process in which RNA molecules inhibit gene expression by neutralising targeted mRNA molecules

[a genetic regulatory system that functions to silence the activity of specific genes]

40
Q

Why did RNAi evolve as a mechanism to block the replication of viruses and to suppress the movements of transposons within the genome?

A

Both of these potentially dangerous processes can involve the formation of double-stranded RNAs

41
Q

Why did RNAi evolve as a mechanism to block the replication of viruses and to suppress the movements of transposons within the genome?

A

Both of these potentially dangerous processes can involve the formation of double-stranded RNAs

42
Q

Describe the formation and mechanism of action of siRNAs (small interfering RNAs)

A

1) Both strands of a double-stranded RNA are cleaved by the endonuclease DICER to form a small siRNA (21-23 nucleotides)
2) the SiRNA becomes associated with a pre-RISC
[In the RNAi pathway, one of the strands of the RNA duplex (PASSENGER STRAND) is cleaved in two and dissociates from the pre-RISC. The other strand (GUIDE STRAND) is incorporated with its ARGONAUTE PARTNER into RISC]
3) The single stranded guide siRNA associated with proteins of the RISC complex binds to a target RNA that has a complementary sequence
4) Target RNA is cleaved at a specific site by the ARGONAUTE PROTEIN and subsequently degraded

43
Q

Describe RISCs in animal cells

A

In animal cells RISCs that contain an siRNA include the Argonaute protein Ago2

44
Q

What might the target RNA be that the single-stranded guide siRNA binds to?

A
  • Viral RNA
  • A transcript from a transposon
  • mRNA
45
Q

What is the clinical importance of siRNA?

A

The ability of siRNA to reduce the expression of any gene of interest just by introducing a short siRNA into a cell suggested the development of THERAPEUTICS (RNAi targets the gene encoding the enzyme)

46
Q

What are microRNAs (miRNAs)?

A

A class of small RNAs that regulate the gene expression

47
Q

What are the properties of miRNAs?

A
  • 21-24 nucleotides in length
  • miRNAs and siRNAs both act in POSTTRANSCRIPTIONAL RNA SILENCING PATHWAYS
  • Regulate gene expression
  • Regulation of apoptotic pathways
48
Q

What is the difference between siRNAs and miRNAs?

A

siRNAs serve primarily to maintain the integrity of the genome whereas miRNAs serve primarily to regulate gene expression

49
Q

What were the first miRNA associated with apoptosis?

A

miR-14 and bantam which were discovered in Drasophila

MOLECULAR BIOLOGY II (14 decks)

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