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BIO230 Labs > Lab 3 > Flashcards

Lab 3 Flashcards

1
Q

What are the classes of RNA in bacterial and eukaryotic cells?

A
  • Ribosomal RNA (rRNA)
  • Transfer RNA (tRNA)
  • Messenger RNA (mRNA)
    Each plays a critical role in protein synthesis
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2
Q

What do mRNAs do?

A

they transfer the information from DNA to the cell machinery that produces proteins. These molecules direct the synthesis of proteins and are the most heterogenous of the RNAs in terms of base sequence and size

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3
Q

What is RNA itself?

A

the final product of the genes coding for the other types of RNA, and are therefore known as noncoding RNAs.

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4
Q

What do tRNAs do?

A

they are essential for translation. They transfer amino acids during protein synthesis.

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5
Q

What do rRNAs do?

A

they combine with proteins and enzymes in the cytoplasm to form ribosomes, which act as the site of protein synthesis.

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6
Q

What do the tRNA and ribosomes do?

A

together, they decode the mRNA message.

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7
Q

What do prokaryotic and eukaryotic ribosomes have in common?

A

they have similar structures and functions, each composed of one large and one small subunit that join to form a complete ribosome with a mass of several million daltons.

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8
Q

What does the small ribosomal subunit do?

A

it provides the framework on which the tRNAs are accurately matched to the codons of the mRNA

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9
Q

What does the large ribosomal subunit do?

A

it catalyzes the formation of the peptide bonds that link the amino acids into a polypeptide chain

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10
Q

How do eukaryotes differ from prokaryotes in terms of RNA?

A

anywhere from 50 to 5,000 sets of rRNA genes and as many as 10 million ribosomes may be present in a single cell

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11
Q

How do prokaryotes differ from eukaryotes in terms of RNA?

A

generally have fewer sets of rRNA genes and ribosomes per cell

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12
Q

How many rRNA genes and ribosomes does E. Coli have?

A

7 copies of the rRNA genes synthesize about 15,000 ribosomes per cell

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13
Q

What is the most abundant RNA form in cells?

A

the most abundant and most stable is rRNA
- demonstrated by extracting total cellular RNA and separating based on size

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14
Q

How can RNA be separated from a cell?

A

sedimentation through a sucrose gradient or by gel electrophoresis.

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15
Q

How are RNA subunits named?

A

rate of sedimentation, measured in Svedberg units [S]

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16
Q

What is the most detectable RNA on a gradient or gel?

A
  • rRNA (70-80%)
    -tRNA (10-20%)
    -mRNA (less than 10%)
17
Q

Why is RNA isolated?

A

to perform many fundamental molecular biology experiments, the first and most crucial step is isolation high quality, intact RNA

18
Q

What kinds of experiments need to isolate RNA?

A

-RT-PCR
-nuclease protection assays
- in vitro translation
- cDNA library construction, micro analysis
- molecular cloning
- RNA-seq

19
Q

What does electrophoresis do?

A

used to separate molecules via an electric current

20
Q

How does electrophoresis work?

A
  • molecules to be separated are placed in wells of an agarose gel that is submerged below the surface of a buffer
  • molecules will move in the electrical field according to their charge
21
Q

Where will molecules move based on their charge?

A

pos. charged molecules move toward the neg. charged electrode (cathode)
neg. charge molecules move towards the pos. charged electrode (anode)

22
Q

Where would DNA and RNA move in an electric field?

A

they carry a neg. charge (backbone) and move towards the pos. electrode

23
Q

What does the rate of migration of a molecule depend on?

A

2 factors:
- molecular structure
- concentration of agarose

24
Q

How can DNA and RNA be separated?

A

the size fragments of the samples are loaded onto an agarose gel, which is comprised of a network of pores through which the nucleic acids must travel to reach the pos. electrode.

25
Q

How do molecules travel through agarose gel?

A

the smaller the fragments, the faster it can travel through the gel

26
Q

What does changing the concentration of agarose in the gel do?

A

it can be varied to improve the resolution of fragments

27
Q

What does a higher concentration of agarose in the gel do?

A

it can be used to increase the separation of smaller DNA fragments.

28
Q

What does a lower concentration of agarose in the gel do?

A

it can be used to separate large fragments

29
Q

What does the success or failure of extracting RNA depend on?

A

your ability to avoid and overcome RNase activity which will destroy your sample (denature it) and it’s found on everything like skin

30
Q

Why is E. Coli a model organism?

A
  • relative genetic simplicity, rapid generation time, ease of working with
31
Q

What is E. coli used to investigate?

A
  • DNA replication
  • gene expression
  • protein synthesis
32
Q

What is EDTA?

A

it is a chelator, which has a high affinity for Mg2+. When divalent cations are all bound to chelator, RNases requiring these ions become inactive. THis protects the RNA from digestion during its isolation.

33
Q

What does lysozyme do?

A

Disrupts the cell wall of the bacteria

34
Q

What does SDS do?

A

it’s a strong ionic detergent that solubilizes the plasma membrane. It’s also a strong protein denaturant

35
Q

What does potassium acetate do?

A

forms ionic bonds to produce insoluble potassium dodecyl sulphate. All molecules bound to SDS before now precipitate from the solution with the PDS

36
Q

Why is NaCl used?

A

high salt concentrations are used to help precipitate RNA. The Na+ also neutralizes the neg. charge on the RNA backbone which decreases the solubility in water.

37
Q

What does ethanol do?

A

It further reduces the solubility of the RNA in the aqueous solution.

38
Q

How are ribosomal RNA molecules produced in both cell types?

A

as large primary transcripts (pre-ribosomal RNA) that require subsequent processing

39
Q

What is the primary transcripts of prokaryotic rRNA molecules?

A

they are about 30S in size and contain one copy of each
- 16S
- 23S
-5S
- several tRNA precursors

BIO230 Labs (4 decks)

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