MCBG Session 13 - Gene Expression

Question 1

What is the central dogma of biology?
What are the 2 processes in this dogma called?
Where do these processes occur within the cell?

Answer 1

DNA –> RNA –> Protein
DNA –> RNA = Transcription (reading the code)
RNA –> Protein = Translation (code transformed)
Transcription = nucleus. Transcription = cytoplasm (RER)

Question 2

What are the 3 regions on DNA involved in transcription?

What terminology is used for directions of these regions?

Answer 2

1) Promoter region (where initation of transcription occurs)
2) Coding region - region where code is copied
3) Terminator region (where termination of transcription occurs)

Upstream & Downstream - e.g.: coding region is always upstream of promoter region.

Question 3

Does a gene code for 1 protein, 1 polypeptide or 1 functional unit? Explain why.

Answer 3

• Doesn’t code for just one protein, a gene can code for multiple proteins, e.g.: Hb has 2 genes.
• Doesn’t code for just one polypeptide, as not all genes are expressed as polypeptides and not all RNA molecules are translated.
• 1 gene does code for 1 functional unit - either a sequence of AA’s in a polypeptide or a sequence of nucleotides in untranslated RNA.

Question 4

What are the main differences between DNA and RNA.

Answer 4

1) RNA has hydroxyl group of C2 (DNA has H removed)
2) RNA contains uracil (U) instead of thymine (T)
3) RNA molecule are single stranded (DNA is double)
4) RNA can form stem loops and other complex 3D structures with non-standard base pairings.

Question 5

How much of total RNA does rRNA make up?
How many kinds and copies or rRNA are there?
How well conserved is rRNA?

Answer 5

• 80% of total RNA
• Few kinds, but many copies of rRNA
• Very well/highly conserved

Question 6

How much of total RNA does tRNA make up?
How many kinds and copies of tRNA are there?
What is each tRNA dedicated to?

Answer 6

• 15% of total RNA
• Roughly 100 kinds, many copies (generally small)
• Each tRNA is dedicated (cognate) to one of the 20 AA’s

Question 7

How much of total RNA does mRNA make up?

How many kinds and copies of mRNA are there?

Answer 7

• 2-5% of RNA
• 100,000’s of kinds, very few copies

NB: there are many other types of RNA, e.g.: SiRNA which have important functions within the cell.

Question 8

What are the 3 things a cell requires for transcription to occur and what are the 3 stages?

Answer 8

1) A template - DNA code
2) An enzyme - RNA polymerase
3) Substrates - NTP’s (nucleotide triphosphates)

Initation –> Elongation –> Termination

Question 9

What drives the reaction of transcription?

What direction does synthesis of RNA occur in?

Answer 9

• Hydrolysis of PPi by pyrophosphates

- Synthesis from 5’ to 3’

Question 10

Which RNA polymerase enzymes synthesis rRNA, mRNA and tRNA?

Answer 10

rRNA = RNA polymerase 1
mRNA = RNA polymerase 2
tRNA = RNA polymerase 3

Question 11

Which subunit on RNA polymerase is required for specific DNA binding on promoter?

Answer 11

Sigma subunit - makes RNA specific, otherwise would be made at random.

Question 12

What occurs during initiation of transcription?

Answer 12

• Transcription factor (TF) binds to promoter region
• Specifically to TATA box (specific sequence recognised by TF’s)
• More TF’s bind to sequences upstream of TATA
• RNA polymerase is recruited

Question 13

What occurs during elongation of transcrption?

Answer 13

• DNA helix unwound into single strand
• Formation of transcription bubble (17bp)
• Template strand read 3’ to 5’, RNA synthesis occurs 3’ to 5’
• As transcription bubble advances, DNA helix reforms behind it

Question 14

What is the transcribed and non-transcribed strand of DNA in transcription called?

Answer 14

Non-transcribed = coding strand
Transcribed = template strand

Question 15

Transcription termination is sequence-dependent, what does this result in the production of?
What is required to produce the mature version?

Answer 15

• Primary RNA

- RNA processing needed to produce mature RNA

Question 16

What are the main events that occur during RNA processing?

Answer 16

1) Post-translational modification - e.g.: chemical modifications and cleavage (in rRNA and tRNA)
2) 5’ cap added (5’ to 5’) which provides protection
3) 3’ polyA tail cap added for protection and regulation
(caps added in mRNA only)
4) Splicing - introns removed, exons joined up by spliceosomes

Question 17

Therefore, what regions does mature mRNA contain?

Answer 17

• Open reading frame (ORF)
• 5’ untranslated region (UTR)
• 3’ UTR

Question 18

What is the difference between ribosomes in prokaryotes and eukaryotes?

Answer 18

Prokaryotes = 3 x rRNA’s + 56 proteins. 30s and 50s subunit combine to form 70s ribosome

Eukaryotes = 4 x rRNA’s + 82 proteins. 40s and 60s subunits combine to form 80s ribosome (larger)

Question 19

What is the template, enzyme and substrate required for translation of mRNA?

Answer 19

Template = mRNA
Enzyme = rRNA in ribosomes
Substrate = Activated amino acids

Question 20

How does tRNA bind an AA at its AA attachment site?

Answer 20

Aminoacyl tRNA synthetase binds and “charges” an amino acid which allows for specific binding of AA’s to specific tRNA molecules

Question 21

What is required in order for translation to occur?

Answer 21

• Mature mRNA
• Ribosomes
• tRNA’s charged with activated AA’s
• Initiation factors (IF), elongation factors (EF), release factors (RF) and energy (ATP/GTP)

Question 22

What occurs during initiation phase of translatiton?

Answer 22

• tRNA recognises start codon (AUG - Methionine)
• This is done by anticodon of initiation complex
• IF’s involved
• Recruitment of large ribosomal sub-unit (50s)
• Assembly of functional ribosome

Question 23

What occurs during the elongation phase of translation?

Answer 23

• Binding of aminoacyl-tRNA on A-site
• Peptide bond formation catalysed by peptidyltransferase
• Growing peptide is pushed into exit tunnel (EF involved)

Question 24

What occurs during termination phase of translation?

Answer 24

• Recondition of stop codon
• Binding of RF (recognises stop codon)
• Hydrolysis of resulting free peptide (through tunnel)
• Dissociation of ribosome into sub-units