Gene organisation, transcription and regulation

Question 1

Features of DNA

Answer 1

• Deoxyribose and phosphate backbone
• Adenine, guanine, cytosine, thymine bases
• C-H bonds make deoxyribose sugar less reactive than ribose
• Stable in alkaline conditions
• Minor grooves make it harder for enzymes to attack DNA

Question 2

Features of RNA

Answer 2

• Ribose and phosphate backbone
• Adenine, guanine, cytosine, uracil bases
• C-OH bonds make ribose more reactive than deoxyribose
• Unstable in alkaline conditions
• Has larger grooves so more easily attacked by enzymes

Question 3

Transcription process

Answer 3

• DNA strand unwinds
• Complementary ribonucleotides placed and joined together by phosphodiester bonds
• RNA chain grows one base at a time in 5’ to 3’ direction
• RNA chain is completed and removed from DNA strand
• DNA strand rewinds

Question 4

The major classes of RNA and their respective RNA polymerases

Answer 4

rRNA - ribosomal RNA = RNA polymerase 1
tRNA - transfer RNA = RNA polymerase 2
mRNA - messenger RNA = RNA polymerase 3

Question 5

Gene promoter definition

Answer 5

The DNA sequence at which RNA polymerase binds

Question 6

Transcription factor definition

Answer 6

A protein that controls the rate of transcription by suppression or activation

Question 7

Process of Basal Transcription Complex assembly

Answer 7

• Transcription factor (TF) IID binds to TATA DNA sequence
• TF IIA and TF IIB bind to TF IID
• RNA polymerase 2 with TF IIF attached bind.
• TF IIJ, IIE, IIH bind to RNA polymerase 2
  (See diagram in notes)
  TF IID, TF IIA and TF IIB, RNA polymerase 2 and TF IIF, TF IIE, TF IIH, TFIIJ.

Question 8

Role of basal transcription complex (BTC)

Answer 8

Allows RNA polymerase 2 to be phosphorylated and carry out transcription
- with no other TFs present, this complex produces a low (basal) level of transcription

Question 9

Introns definition

Answer 9

Sequences in the gene which are transcribed but edited out of the final mRNA

Question 10

Exons definition

Answer 10

Segments of the gene which contain sequences that form part of the final mRNA

Question 11

Sequence of events in pre-mRNA splicing

Answer 11

• U1 binds to splice donor sequence
• U2, U4, U5, U6 all bind and form the splicing complex (spliceosome) which cleaves splice donor sequence
• An “A” residue in the intron is used as the branch point in an intermediate step.
• The “branch” results from cleavage of the phosphodiester bond at the start of the intron
• End of cleaved intron forms bond with brnachpoint “A”
• End of intron is cleaved and intron removed as a “lariat” structure
• The exposed ends of adjacent exon sequences are joined together (ligated)
  (see diagram in notes)

Question 12

Definition of lariat structure

Answer 12

Cleaved intron

Question 13

Last two nucleotides of exons

Answer 13

AG

Question 14

First two nucleotides of introns

Answer 14

GU

Question 15

Sequence at end of introns

Answer 15

15 pyrimidine nucleotides, any Nucleotide, C, A, G

Pyr15NCAG

Question 16

Sequence of splice donor site

Answer 16

End of exon + start of intron = AGGU

Question 17

Splice acceptor site

Answer 17

End of intron = Pyr15NCAG

Question 18

Composition of the added ‘cap’ to pre-messenger RNA

Answer 18

• Formed by hydrolysis of terminal triphosphate or mRNA to a diphosphate
• Further modified by methylation at N7 position in purine ring to form 7-methylguanylate cap

Question 19

Function of added ‘cap’ to pre-messenger RNA and how it can be targeted by viruses

Answer 19

• Acts to protect mRNA at 5’ end
• Enhances translation of mRNA
• Viruses such as Polio can interfere with recognition of the cap during translation

Question 20

Process of addition of poly-A tail to pre-messenger RNA and its composition

Answer 20

• Polyadenylation
• Poly A tail added one base at a time
• Added 11-30 bases downstream of AAUAAA sequence which is found in ALL mRNAs
• Essentially just made up of a long tail of A’s (AAAA etc)

Question 21

Mechanisms of RNA interference

Answer 21

• Production of antisense RNA to block mRNA translation
• Production of siRNA from dsRNA
• RNA silencing

Question 22

Mechanism of antisense RNA being used to block mRNA translation

Answer 22

• Antisense RNA is the reverse of the mRNA being transcribed

- Binds to pre-transcribed RNA to form dsRNA (double-stranded RNA) which cannot be transcribed

Question 23

Mechanism of dsRNA being used to produce siRNA

Answer 23

• siRNA inhibits RNA that dsRNA is derived from
• Used as a defence mechanism against viral infection
• Small single-stranded RNAs that anneal to viral RNA and degrade them

Question 24

Meaning of dsRNA

Answer 24

Double-Stranded RNA

Question 25

Meaning of siRNA

Answer 25

Small Interfering RNA

Question 26

Process of RNA silencing

Answer 26

- DICER breaks up dsRNA/shRNA into 21-25bp fragments - siRNA guides endonuclease activity to remove on of the siRNA strands called the passenger strand. - Retained strand is antisense to the target strand - Multiprotein RNA-induced silencing complexes (RISCs) are formed - RISCs recognises antisense siRNA that has bound to target strand and cleaves target - Target mRNA now cleaved and unable to be translated

Question 27

What is DICER

Answer 27

An enzyme that cleaves double-stranded RNA to form small double-stranded interfering RNAs (siRNAs)

Question 28

Meaning of shRNA

Answer 28

Short Hairpin RNA, designed to be used in RNA interference

Question 29

What are miRNAs

Answer 29

- MicroRNAs | - Involved in regulating other genes

Question 30

Typical length of a miRNA in nucleotides

Answer 30

18-25 nucleotides

Question 31

miRNA gene regulation mechanisms

Answer 31

- Sequences end up as siRNA that end up in the RISC - Mature miRNAs bind to the 3'-untranslated region of mRNAs and subsequently destabilise them, block their translation or both - Gene knockdown, decreasing the amount of protein created from a gene