Control of Eukaryotic Gene Expression

Question 1

what is the purpose of regulation of gene expression?

Answer 1

to allow cell differentiation

tissue, temporal, spatial specificities: only a fraction of the genome (all 23 chromosomes for humans) in a eukaryotic cell are expressed at any one place or at any one time

Question 2

how does the control of eukaryotic gene expression (EGE) differ from that in prokaryotes?

Answer 2

eukaryotic DNA is organised into nucleosomes: for transcription initiation, genes must be ‘active’ (accessible) to transcription factors

no operons in eukaryotic genes: each gene needs individual regulatory sequences

processes of transcription and translation in eukaryotes separated by nuclear envelope: cannot occur simultaneously, eukaryotic pre-mRNA must be processed and translocated

Question 3

what are the five different levels of control of EGE?

Answer 3

chromatin: histone modification and DNA methylation
transcriptional: initiation of transcription (with control elements and proteins)
post-transcriptional: 5’ 7-methylguanosine cap, splicing, 3’ poly (A) tail
translational: half-life of RNA and initiation of translation
post-translational: biochemical modification and protein degradation

Question 4

why is gene accessibility regulated on a chromatin level, and what are the two methods?

Answer 4

chromatin has “beads (nucleosome: DNA complexed with histone octamer) on a string” appearance
can be organised into euchromatin (diffused) or heterochromatin (highly condensed)
condensation prevents transcription factors and RNA polymerases from gaining access to the promoter of a specific gene

two mechanisms: histone modification and DNA methylation

Question 5

describe how histone modification (acetylation and deacetylation) function in controlling EGE

Answer 5

• acetylation: addition of (negatively-charged) acetyl functional group
  catalysed by histone acetyltransferases (HATs)
  neutralising lysine’s positive charge, reducing affinity for histone complex
  chromatin more diffused / less compact, easier to translate
• deacetylation: removal of acetyl from lysine residues in histone tails
  catalysed by histone deacetylases (HDACs)
  lysine residues regain positive charges, increasing affinity of histone complex for DNA
  chromatin more compact / less diffused, prevents access of transcription factors and RNA polymerase, harder to translate

Question 6

describe how DNA methylation functions in controlling EGE

Answer 6

addition of methyl groups (-CH2) to specific nucleotides after DNA replication (cytosine nucleotides in vertebrate DNA, only sequence of 5’-CG’-3’ called CpG)
catalysed by DNA methyltransferases

CpG dinucleotides cluster to form islands, usually found in promoter regions, preventing transcription due to

1. specific 3D conformation of DNA changed, transcription factors cannot bind to promoter, transcription not initiated
2. methylated DNA recognised by methyl-CpG-binding proteins (MeCPs) that recruit other proteins like histone deacetylases (HDACs), histone deacetylation occurs, chromatin more condensed, preventing binding of transcription factors and RNA polymerase, transcription not initiated

Question 7

compare the processes of DNA methylation and histone modification

Answer 7

site: DNA methylation @ CpG islands, histone modification @ histone tails
enzyme: DNA methyltransferases (DNA methylation), histone acetyltransferases (HATs, for acetylation), histone deacetylases (HDACs, for deacetylation)
outcome: DNA methylation and deacetylation down-regulate (increases) transcription, acetylation (decreases) up-regulates transcription

Question 8

what does control of transcription initiation determine?

Answer 8

whether or not genes are expressed
quantity of encoded mRNAs
(consequently) quantity of proteins produced

Question 9

what is required for transcription initiation, and how is the maximum transcription rate of a gene achieved?

Answer 9

initiation: general transcription factors and RNA polymerase bind to promoter to form transcription initiation complex (TIC)
maximum: interaction of specific transcription factors and distal control elements (enhancers or silencers) with general transcription factors and RNA polymerase to form stable TIC

Question 10

what are the control elements for transcription, and how do the particular combinations result in specificity of transcription?

Answer 10

control elements: promoter, proximal control elements, distal control elements (enhancers and silencers) bind different transcription factors (proteins, including activators and repressors)

particular combinations specific to each gene, causing different transcription rates…
in different cell types = spatial specificity
at different developmental stages = temporal specificity

Question 11

define a transcription factor

Answer 11

regulatory protein that binds to DNA and affects transcription of genes

Question 12

what are the general properties of specific transcription factors?

Answer 12

mediate response to stimuli that signal one or more genes should be activated or repressed
recognise and bind to enhancers or silencers
interact with components of transcription machinery (directly or indirectly)
two binding domains: DNA binding and protein (aka, activation) domains for protein-protein interactions
two groups: activators (bind to enhancers to increase rate) and repressors (bind to silencers to decrease rate)

Question 13

describe the series of events leading to transcription initiation (wrt transcription factors)

Answer 13

• activators (proteins) bind their respective enhancers (non-coding DNA sequence)
• GTFs bind to promoter, mediate binding of RNA polymerase, forming transcription initiation complex (rate of transcription is “basal”, slow)
• DNA-bending protein causes DNA looping: activators bound to enhancers (distal promoter elements) far upstream or downstream are brought close to promoter
• activators interact with mediator proteins (adaptor molecules), facilitating interaction of activators with GTF and RNA polymerase
• improved recruitment of GTF and RNA polymerase to promoter, stabilising TIC
• activator properly positions TIC on promoter
• ROR increased

Question 14

what does the precise temporal/spatial control of transcription largely depend on in eukaryotes?

Answer 14

binding of activators to respective enhancers
particular combination of enhancers associated with gene can only activate transcription when appropriate activators are present during precise developmental timing or in specific cell type

Question 15

what are the six different mechanisms of transcription repression by repressor proteins?

Answer 15

1. competitive DNA binding: activator and repressor proteins compete for same binding site of DNA sequence
2. masking activation surface: both bind DNA, but repressor prevents activator from interacting with GTFs (other proteins)
3. repressor blocks assembly of GTFs: by binding to GTF itself
4. repressor recruits chromatin remodeling complex: returns nucleosomal state of promoter region to pre-transcriptional form
5. repressor attracts histone deacetylase to promoter: reverses histone acetylation, repressing transcription initiation
6. attracts histone methyltransferase: add methyl groups of histones, methylated histones bound by proteins that maintain chromatin in transcriptionally silent form

Question 16

how is mRNA spliced?

Answer 16

1. cleavage at 5’ splice site, joining intron to a branch point within the intron, yielding lariat-like intermediate (loop)
2. cleavage at 3’ splice site, simultaneous ligation (joined by phosphodiester bond) of exons, excision of intron as lariat-like structure,. DNA sequences at 5’ and 3’ ends of intron serve as recognition sites for spliceosomes to bind

Question 17

what is alternative splicing, and what is its function?

Answer 17

use of different splice sites to be joined in different combinations (same pre-mRNA yields different mature mRNA yields different proteins)

substantial proportion of higher eukaryotic genes synthesise different proteins from one gene in this way

Question 18

what is the relationship between stability of mRNA and duration for which translation occurs, and compare the half-lives of eukaryotic and prokaryotic cells

Answer 18

more stable = more translation
mRNAs in eukaryotic cells more stable or longer half-lives (time required for 50% of initial amount of RNA to be degraded) than that of prokaryotic cells

Question 19

what is the stability of mRNA affected by, and what are the mechanisms for the decay of eukaryotic mRNA?

Answer 19

stability affected: length of poly(A) tail and stabilising or destabilising sequences in 3’ UTR

decay– both start with gradual shortening of poly(A) tail by an exonuclease (begins once mRNA reaches cytosol). at a critical length (around 25 nucleotides for humans), two pathways diverge

1. 5’ cap removed in ‘decapping’, “exposed” mRNA rapidly degraded from 5’ end
2. mRNA continues degradation from 3’ end, through poly(A) tail into coding sequences
   * both processes can occur together)

Question 20

what is the specialised mechanism that degrades mRNAs, and how does it work?

Answer 20

endonuclease (specific nucleases that cleave mRNA internally) removes both 5’ methylguanosine cap and 3’ poly(A) tail, rapidly degrading DNA
mRNA destroyed this way carry specific nucleotide sequences, often for these endonucleases
very simple to tightly regulate stability of these mRNAs by blocking or exposing endonuclease site in response to extracellular signals

Question 21

what is cytoplasmic poly(A) tail addition, and what does it do?

Answer 21

modifications made to specific mRNAs in cytoplasm to promote translation, by lengthening poly(A) tail (eg. in oocytes, unfertilised egg, when they mature)

Question 22

what are eukaryotic initiation factors and translation repressors, and how can they control EGE?

Answer 22

eIFs: scan the mRNA for the start codon AUG, locating the binding site of initiator tRNA to the AUG codon and forming TIC at 5’ mRNA region
- varying the abundance and activity of these factors to affect rate of translational initiation, global effect on overall translational activity (not specific)

translational repressors: bind to various regions of mRNA (usually 5’ and 3’ UTRs), interfere with initiation of translation by blocking the attachment of ribosomes or other translation initiation factors

Question 23

what are the two mechanisms of translation initiation at the different alternative translation initiation sites?

Answer 23

1. use of subsequent AUG (start codon) for translation initiation: “leaky scanning” could lead to small ribosomal subunit skipping first AUG codon, producing proteins that vary in their N-terminal sequence
2. initiation of translation in middle of mRNA: internal ribosome entry site (IRES, specialised nucleotide sequence) allows for translation initiation in the middle of an mRNA sequence in a cap-independent manner (since the formation of a TIC typically requires 5’ cap recognition), producing protein with different primary structure
   * seen mainly in viruses (refer to PGE)

Question 24

what is RNA interference, which type of RNA is involved in this, and what is its function?

Answer 24

• miRNA do RNA-RNA base-pairing w mRNA, inhibiting transcription and tagging for destruction

Question 25

describe the action of miRNA in the control of EGE

Answer 25

1. single-stranded RNA transcripts (synthesised by transcribing miRNA-coding genes) fold back on themselves, forming (double-stranded) hairpin structure held together by hydrogen bonds
2. processed by Dicer (enzyme), cuts double-stranded ‘hairpin’ RNA into fragments
3. one strand of fragment degraded by RNA-inducing silencing complex (RISC, enzyme), remaining strand binds to RISC, forming miRNA-protein complex
4. single-stranded miRNA binds to mRNA molecules with complementary sequence
5. miRNA-protein (RISC) complex inhibits translation, blocking formation of TIC or mRNA degredation

Question 26

how does the rate of protein activation and breakdown control EGE?

Answer 26

some polypeptides require post-translational modifications: rate of alterations controls rate of functional (active) protein formation

proteins have limited lifespan: some proteins that trigger metabolic changes in cells are broken down within a few minutes or hours, and regulation allows cells to

• adjust the kinds and amounts of proteins in response to stimuli
• maintain proteins in working order