control of eukaryotic gene expression

Question 1

other than to compact DNA into a form that fits into the nucleus, why is DNA condensed into chromatin?

Answer 1

condensation prevents transcription factors and RNA polymerase from gaining access to the promoter of a specific gene, thus inactivating transcription of that gene

Question 2

what are characteristics of histone tails?

Answer 2

1. histone tails are rich in lysine residues, which are positively charged
2. histone tails interact strongly with the negatively-charged phosphate groups of the DNA backbone and increases the affinity of DNA for the nucleosome surfacae

Question 3

what are the 2 chemical modifications to histones and to DNA that can control gene expression?

Answer 3

1. histone modification
2. DNA methylation

Question 4

what are histone tails?

Answer 4

the N-terminus of each histone that protrudes from the nucleosome

Question 5

what are the 2 ways histones can be modified?

Answer 5

acetylation & deacetylation.
the tightness of DNA winding around the histones is altered, altering the ease of transcription initiation as well

Question 6

describe histone acetylation.

Answer 6

1. positively-charged lysine residues in the histone tails can be acetylated by histone acetlytransferases (HATs)
2. when lysines are acetylated, their positive charges are neutralised, and the affinity of the histone complex for DNA is reduced
3. chromatin becomes more diffused/less compact, so control regions of genes would be exposed to transcription factors & RNA polymerase, increasing transcription

in short: acetylation -> makes chromatin less compact -> increased transcription

Question 7

describe histone deacetylation.

Answer 7

1. histone deacetylases (HDACs) catalyse the deacetylation of acetylated lysine residues in histone tails
2. lysine residues regain their positive charges, resulting in an increase in the affinity of the histone complex for DNA
3. chromatin becomes more compact and prevents the access of transcription factors & RNA polymerase to the control regions of genes.

in short: deacetylation -> makes chromatin more compact -> less transcription

Question 8

what is DNA methylation?

Answer 8

the addition of methyl groups to specific nucleotides after DNA replication.
restricted to cytosine nucleotides in the sequence 5’-CG-3’ (aka CpG nucleotides). CpG dinucleotides are clustered to form CpG islands, which are usually found in the promoter regions of genes -> methylation of cytosines within a gene’s promoter sequence prevents transcription of the gene
catalysed by DNA methyltransferases.

Question 9

describe how DNA methylation represses gene expression

Answer 9

1. methylation changes the 3D conformation of DNA and prevents binding of transcription factors to the promoter, preventing transcription initiation
2. methylated DNA serves as recognition signals for methyl-CpG-binding proteins that recruit other proteins like histone deacetylases (HDACs). HDACs makes chromatin in the CpG region more condensed which prevents the binding of transcription factors & RNA polymerase, preventing transcription initiation

Question 10

what is a transcription factor?

Answer 10

a transcription factor is a regulatory protein that binds to DNA and affects transcription of genes

besides general transcription factors and RNA polymerase, specific transcription factors (activators & repressors) affect the expression of certain genes in response to specific signals.

Question 11

what are general properties of specific transcription factors?

Answer 11

• they mediate response to a stimulus, which signals that one or more genes should be switched on or off
• they recognise and bind to enhancers or silencers (DNA sequence)
• they interact indirectly/directly with components of the transcription machinery
• they contain 2 binding domains in their structures - the DNA binding domain (that binds to DNA) and one or more protein binding domains (that bind other regulatory proteins/components of the transcription machinery)

Question 12

what are activators and repressors

Answer 12

• activators (proteins) bind to enhancers (DNA sequence), triggering a series of interactions that result in an increased rate of transcription
• repressors (proteins) bind to silencers (DNA sequence), triggering a series of interactions tha result in a decreased rate of transcription

Question 13

how is rate of transcription increased begin?

activator proteins

Answer 13

1. activator bind to their respective enhancers
2. general transcription factors bind to the promoter and mediate the binding of RNA polymerase, forming a transcription initiation complex. the rate of transcription is basal
3. DNA-bending protein causes the looping of DNA, which allows activator bound to enhancers located far upstream or downstream to be brought closer to the promoter
4. activators interact wih mediator proteins, which serve as adaptor molecules and facilitate the interaction of the activator with general transcription factors and RNA polymerase -> improved recruitment of general transcription factors and RNA polymerase to the promoter, forming a stable transcription initiation complex. activators also facilitate the proper positioning of the transcription initiation complex, initiating transcription -> rate of transcription is increased.

Question 14

how do repressor proteins work?

dn memo, js read thru

Answer 14

1. activator proteins and repressor proteins compete for binding to the same regulatory DNA sequence
2. both proteins bind DNA, but the repressor prevents the activator from interacting with the general transcription factors
3. the repressor blocks assembly of the general transcription factors
4. a chromatin remodelling complex is recruited by the repressor, which returns the nucleosomal state of the promoter region to its pre-transcriptional form (GTF cannot access)
5. the repressor attracts a histone deacetylase to the promoter, which causes histone deacetylation, repressing transcription initiation
6. the repressor attracts a histone methyl transferase, which modifies certain positions on the histones by attaching methyl groups, and the methylated histones are bound by proteins that maintain the chromatin in a transcriptionally silent form

Question 15

how is mRNA spliced?

Answer 15

1. the 5’ splice site is cleaved and the intron is joined to a branch point within the intron.
2. the 3’ splice site is cleaved, and the exons are simultaneously ligated. DNA sequences at the 5’ and 3’ ends of an intron serve as recognition sites for spliceosomes to bind

Question 16

what is stability of mRNA and what is it affected by?

Answer 16

stability of mRNA determines the duration for which translation can occur. (less stable = less time available for it to be translated)
stability of mRNA is affected by:
1. length of poly(A) tail. mRNA with longer poly(A) tails tend to be more stable
2. stabilising/destabilising sequences in the 3’ UTR. these sequences may contain binding sites for specific proteins that increase or decrease the rate of poly-A tail shortening

Question 17

what is alternative splicing and why is it good?

Answer 17

alternative splicing is the use of different splice sites, which allows exons to be joined together in different combinations, producing different mature mRNAs which generate different proteins.
results in greater gene diversity

Question 18

how does eukaryotic mRNA decay?

Answer 18

• the poly(A) tail is gradually shortened by exonuclease. once the poly(A) tail is reduced to a critical length, 2 possible pathways occur.
• pathway 1: the 5’ cap is removed, and the ‘exposed’ mRNA is rapidly degraded from its 5’ end
• pathway 2: the mRNA continues to be decayed from the 3’ end, through the poly(A) tail, into the coding sequence
• some mRNAs can be degraded by a mechanism that uses endonuclease to decap one end and removes the poly(A) tail from the other, such that both halves are rapidly degraded (ggwp)

Question 19

how do translational repressors work?

Answer 19

translational repressors decrease the rate of translational initiation.
these repressors bind to various regions of the mRNA molecule and interfere with the initiation of translation by blocking the attaachment of ribosomes or other translation initiation factors

Question 20

what are alternative translation initiation sites?

Answer 20

use of 2nd or subsequent AUG for translation initiation
- the small ribosomal unit could skip the first AUG codon and use the 2nd or subsequent one
- this is known as ‘leaky scanning’ and results in proteins that vary in their N-terminal sequence

initiation of translation in the middle of mRNA
- translation usually begins at the 5’ end of the mRNA molecule, since 5’ cap recognion is required for the assembly of the initiation complex
- the internal ribosome entry site (IRES) is a specialised nucleotide sequence that allows for translation initiation in the middle of an mRNA sequence in a cap-independent manner, since the need for a 5’ cap structure is bypassed
- a protein with a different primary structure is produced with this mechanism

Question 21

how do microRNAs/miRNAs work?

Answer 21

1. RNA transcripts fold back on themselves, forming a hairpin structure held together by hydrogen bonds
2. they are processed by an enzyme called dicer, which cuts the double-stranded RNA ino smaller fragments
3. one strand of the double-stranded RNA fragment is degraded by a protein complex known as RNA-inducing silencing complex (RISC). the remaining strand binds to RISC to form miRNA-protein complex
4. the miRNA strand then binds to mRNA molecules that have the complementary sequence
5. the miRNA-protein complex then inhibits translation by blocking formation of te translation initiation complex or degradation of the mRNA