Control

Question 1

Gene regulation

Answer 1

Wide range of mechanisms used by cells to ↑/↓ the production of specific gene products → can either be proteins or RNA

Question 2

Constitutively expressed genes

Answer 2

Proteins that are synthesised continuously at a fixed rate

Question 3

Purpose of gene expression regulation

Answer 3

• Cellular differentiation: regulation of genes → production of different proteins → cells have different ultrastructures that their functions i.e. in a multicellular organism, all somatic cells carry identical genes but cells show a wide variation in structure and function
• Adapt to changes: vary according to circumstances and demand
• Conserve resources (transcriptional level control predominates as it is the most efficient mechanism with minimal wastage, esp in prokaryotes)
• More varied proteome despite limited genome size

Question 4

Control at genomic level (4)

Answer 4

1. Chromatin remodelling complex
2. DNA methylation
3. Histone acetylation/deacetylation
4. Gene amplification

Question 5

Organisation of chromatin structure

Answer 5

• Pact DNA into compact form and regulate gene expression
• Heterochromatin → highly compacted → DNA winds more tightly around histones → limits access of RNA pol and GTF to promoters → prevents formation of TIC → silences genes
• Euchromatin → less compacted → DNA winds less tightly → allows access of RNA pol and GTF to promoters → allows formation of TIC → transcriptionally active

Question 6

1. Chromatin remodelling complex

Answer 6

• Protein complexes that alter structure of nucleosomes temporarily:
  a) DNA less tightly bound to histones → promotes transcription → promotes assembly of TIC
  b) DNA more tightly coiled around histones → blocks transcription → prevents assembly of TIC

Question 7

2. DNA methylation

Answer 7

• Adds methyl group to selected cytosine (C) nucleotides located in CG sequences to prevent transcription → gene silencing
  a) Block binding of GTF → prevent assembly of TIC
  b) Recruit DNA-binding proteins (repressors, histone deacetylase, repressive chromatin remodeling complexes) → condense chromatin

Question 8

3. Histone acetylation/deacetylation

Answer 8

Acetylation, catalysed by histone acetyltransferase

• Adds acetyl groups to lysine residues
• Removes positive charges on histones → decrease electrostatic interactions between -vely charged DNA and histones
• Tight binding loosened → promoter region more accessible to RNA pol and GTF → form TIC → promote transcription

Deacetylation, catalysed by histone deacetylase

• Removes acetyl groups
• Restore +ve charge on histone → restore tighter interaction
• Promoter region more accessible to RNA pol and GTF → form TIC → promote transcription

Question 9

4. Gene amplification

Answer 9

• Replication of specific gene multiple times → create more copies
• Gene of interest exists in high copy number → ↑ copies of mRNA → ↑ copies of required protein

Question 10

Control at transcriptional level (3)

Answer 10

• Control elements → non-coding DNA segments that transcription factors bind to
• Transcription factors → gene regulatory proteins
• Proximal → upstream of transcriptional start site → GTF
  1. Promoters
• Distal → thousands of nucleotides up/downstream, or even within intron → STF
  2. Enhancers
  3. Silencers

Question 11

1. Promoter

Answer 11

• Recognition and binding site for GTFs which then recruits RNA form TIC which initiates transcription
• TATA box (-25) determines precise location of transcription start site
• CAAT (-75) and GC (-90) boxes not always present, can have multiple copies
• Improve efficiency of promoter by helping to recruit GTF and RNA pol

Question 12

2. Enhancers

Answer 12

• Positive regulatory elements → upregulation
• Allows activators (STF) to bind → promote assembly of TIC → ↑ frequency of transcription

a) Bending of spacer DNA allows interaction of activators with RNA pol and/or GTFs at the promoter
b) May recruit histone acetyltransferase and chromatin remodeling complex to decondense chromatin → ↑ accessibility of promoter to GTFs and RNA pol

Question 13

3. Silencers

Answer 13

• Negative regulatory elements → downregulation
• Allows repressors (STF) to bind → inhibit assembly of TIC → ↓ frequency of transcription

a) Interfere with action of activator:
i) Competitive DNA binding
ii) Masking activation surface
iii) Direct interaction with GTFs

b) May recruit histone deacetylase and repressible chromatin remodelling complex to condense chromatin → ↓ accessibility of promoter to GTFs and RNA pol

Question 14

Control at post-transcriptional level

Answer 14

• Pre-mRNA → mature mRNA
  1. Capping at 5’ end
  2. Splicing of pre-mRNA
  3. Adding poly-A tail to 3’ end (polyadenylation)

Question 15

1. Capping at 5’ end of pre-mRNA

Answer 15

• Addition of a 7-methylguanosine nucleotide
• Occurs shortly after transcription begins i.e. occurs co-transcriptionally

1. helps the cell to recognise mRNA (amongst other RNAs) so that subsequent steps such as splicing and polyadenylation can occur
2. signal to export mRNA out of nucleus
3. protects the growing pre-mRNA chain from degradation by ribonucleases
4. promotes initiation of translation as it is recognized by translation initiation factors that help recruit mRNA to small ribosomal subunit

Question 16

2. Splicing of pre-mRNA

Answer 16

• Introns excised, exons joined together
• Spliceosome → snRNA-protein complex
• Precise intron-exon boundaries
• Produce functional proteins
• Alternative splicing
• Different exons of a single pre- mRNA joined together
• Different mature mRNA
• Different proteins can be produced

Question 17

3. Adding of poly-A tail (polyadenylation)

Answer 17

• When 3’end of pre-mRNA is cleaved by endonucleases to make it shorter
• Poly-A polymerase recognises the polyadenylation signal (AAUAAA)
• Adds a long sequence of adenine nucleotides to 3’ end of the pre-mRNA, forming a poly(A) tail
• Occurs immediately after transcription

1. signal to export mature mRNA out of nucleus
2. protects mature mRNA from degradation by ribonucleases → more proteins can be made
3. Required, together with 5’cap for initiation of translation

Question 18

Control at translational level (3)

Answer 18

1. mRNA half-life/stability
2. Binding of small ribosomal subunit
3. Initiation factors

Question 19

1. mRNA half-life/stability

Answer 19

• Determined by the length of its poly-A tail
• Longer poly-A tail, the longer the mRNA can be used as a template to make proteins.
• Poly-A tail removed by ribonucleases in the 3’ to 5’ direction until a critical length is reached
• Triggers removal of the 5’cap and degradation of mRNA from 5’end too

Question 20

2. Binding of small ribosomal subunit

Answer 20

• Binds to 5’ cap of mRNA
• Can be blocked by translational repressor protein that binds to:
  a) 5’ cap
  b) 5’ untranslated region
  C) 3’ untranslated region → interfere with interaction between 3’ poly-A tail, initiation factors and 5’ cap

Question 21

3. Initiation factors

Answer 21

• Needed to begin protein synthesis → proper positioning of small ribosomal subunit with initiator tRNA on mRNA and recruitment of large ribosomal subunit
• Activation/inactivation by phosphorylation/dephosphorylation

Question 22

Control at post-translational level (3)

Answer 22

1. Covalent modification to form functional proteins
2. Phosphorylation/dephosphorylation to regulate protein activity
3. Protein degradation

Question 23

1. Covalent modification to form functional proteins

Answer 23

• Cleavage or covalent modification
• Glycosylation
• Disulfide bond formation
• Attachment of prosthetic groups
• Occur when polypeptides pass through rER and GA

Question 24

2. Phosphorylation/dephosphorylation to regulate protein activity

Answer 24

• Of translation initiation factors → activate/deactivate protein
• Up/down regulate its activity
• Signal transduction

Question 25

3. Protein degradation

Answer 25

- Determines how long protein remains in cell - Protein degradation by proteasomes - Proteins targetted for degradation are tagged with ubiqutin (by ubiquitin ligase) and then recognised and degraded by the proteasome

Question 26

Control Summary

Answer 26

1. Genomic level a) Chromatin remodelling complex b) DNA methylation c) Histone acetylation/deacetylation 2. Transcriptional level a) Promoters b) Enhancers c) Silencers 3. Post-transcriptional level a) Capping at 5' end b) Splicing of pre-mRNA b) Adding poly-A tail to 3' end (polyadenylation) 4. Translational level a) mRNA stability b) Small ribosomal subunit c) Initiation factors 5. Post-translational level a) Covalent modification b) Phosphorylation/dephosphorylation c) Protein degradation