Nucleic acids 5-7

Question 1

What is non-coding DNA?

Answer 1

Any RNA molecule that is not translated into a protein. Does not include mRNA.

Question 2

How much of non-coding DNA has a function?

Answer 2

20%

Question 3

How much of non-coding DNA has no known function as of yet?

Answer 3

60%

Question 4

What are the four main types of non-coding DNA with known functions?

Answer 4

1) microRNA
2) siRNA (RNAi)
3) piRNA
4) long ncRNA

Question 5

What does microRNA do?

Answer 5

Controls translation of roughly 30% of all genes.

Question 6

What does siRNA do?

Answer 6

Involved in viral defence.

Question 7

What does piRNA do?

Answer 7

Germ cell proliferation.

Question 8

What does long ncRNA do?

Answer 8

Important for X chromosome inactivation.

Question 9

What is X-chromosome inactivation?

Answer 9

Also known as lyonization. When one X chromosome in females is inactivated due to a special conformation (heterochromatin) preventing translation.

Question 10

How long are microRNA strands typically?

Answer 10

18-26 nucleotides. Precursors are between 60 and 80 nucleotides long before exonuclease activity cleaves off the final microRNA strand.

Question 11

How do microRNA work?

Answer 11

Can inhibit mRNA through incomplete base-pairing in certain binding sites, reducing translation of that gene. One microRNA can target several different mRNA binding sites.

Question 12

What is DNA cloning?

Answer 12

Selectively amplifying certain DNA sequences to generate homologous DNA populations. Cell-based or cell-free (PCR).

Question 13

What do restriction endonuclease do?

Answer 13

Cleave DNA at specific recognition sequences (normally 6-8bp palindromic sequences)

Question 14

How is host DNA protected from restriction endonuclease activity?

Answer 14

RE sites are methylated (by methylase) and REs will only cleave unmethylated DNA from invading organisms.

Question 15

What gels are normally used in gel electrophoresis?

Answer 15

Agarose or polyacrylamide.

Question 16

What is nucleic acid hybridisation?

Answer 16

Form of detecting specific nucleic acid sequences, allowing single-stranded DNA or RNA to anneal to form double stranded molecules.

Question 17

How does nucleic acid hybridisation work?

Answer 17

• Probe identifies targets
• Target DNA is immobilised
• Target DNA is hybridised (radioactively/fluorescently)
• DNA is isolated

Carried out 25 degrees below melting temperature of DNA.

Question 18

What does denaturation of DNA depend on?

Answer 18

• Strand length
• Base composition (GC bonds take more energy to break)
• Chemical environment (denaturants and monovalent cations)

Question 19

What enzyme is used in PCR?

Answer 19

taqDNA polymerase (thermostable)

Question 20

Where does translation occur?

Answer 20

Ribosome

Question 21

What is the start codon sequence?

Answer 21

AUG

Question 22

What do tRNA molecules do?

Answer 22

Transport amino acids to the ribosome during translation

Question 23

What bring the amino acid to the tRNA molecule?

Answer 23

Aminoacyl tRNA syntheses (tRNA activating enzymes)

Question 24

What takes place in the invitation stage?

Answer 24

1) Ribosomal subunits disassociate
2) Pre-initiation complex forms (initiation factors bind to 5’ cap and Met-tRNA binds to 40S subunit)
3) mRNA binds to pre-initiation complex
4) 60S subunit binds

Question 25

What takes place during the elongation stage?

Answer 25

1) tRNA molecule binds to A site 2) Catalysis of peptide bond between two amino acids 3) Translocation to P site using energy from GTP

Question 26

What takes place during the termination stage?

Answer 26

1) Recognition of stop codon | 2) Release

Question 27

What does GTP do during translation?

Answer 27

Hydrolysis allows translocation of tRNA molecules along ribosome as well as providing "pauses" to allow for correct base pairing recognition.

Question 28

What are polyribosomes?

Answer 28

Multiple ribosomes working on one strand of mRNA.

Question 29

What can occur in post-translation modification?

Answer 29

Diversity of proteins: - Disulphide bridge formation - Proteolytic cleavage (hormones) - Glycosylation (carbohydrate) - Phosphorylation - Hydroxylation - Acylation and prenylation (addition of lipid groups)

Question 30

What are siRNAs and what is their function?

Answer 30

Short interfering RNAs that combine with mRNA via hybridisation to interfere with the expression of genes and prevent translation of specific mRNA strands.

Question 31

What is RISC?

Answer 31

RNA induced silencing complex - destroys the target mRNA by binding to the hybridised mRNA, causing the transcript to break and preventing translation.

Question 32

Which ribosomal subunit binds to the mRNA first?

Answer 32

Small subunit

Question 33

What are the two classes of antibiotics used to prevent translation of bacteria?

Answer 33

50S and 30S inhibitors

Question 34

How do 50S inhibitors work?

Answer 34

Physically block initiation or translocation of peptides tRNAs by inhibiting peptides transferase.

Question 35

How do 30S inhibitors work?

Answer 35

e.g. streptomycin | Bind to the 16 rRNA subunit and interfere with peptides-tRNA binding.