3.1 Flashcards
1
Q
what is a gene
A
inherited unit of information that on the gross level determines phenotype (morphological characteristics) and on the molecular level determines amino acid sequence of a polypeptide / RNA
1
Q
types of mutations
A
substitution
deletion - frameshift
insertion - frameshift
duplication - frameshift
2
Q
types of substitution
A
missense - change in amino acid
nonsense - premature stop codon
3
Q
types of swaps of amino acids
A
transitions - pyrimidine to pyrimidine or purine to purine
transversions - pyrimidine to purine + vice versa
4
Q
what is a silent mutation
A
when the point mutation in a base causes the same amino acid to still be coded for = does not effect polypeptide
5
Q
how do mutations occur
A
spontaneous or induced via mutagens
6
Q
w - hwo do mutations occur…
A
7
Q
w
A
8
Q
w
A
9
Q
w
A
10
Q
w
A
11
Q
w
A
12
Q
w
A
13
Q
w
A
14
Q
w
A
15
Q
what is the molecular structure of DNA
A
double helix
two antiparallel strands
made up of nucleotides - deoxyribose sugar + phosphate + nitrogenous base
AT - 2 - H bonds
CG - 3 - H bonds
Sugar phosphate back bone - phosphodiester bonds
major + minor grooves
5’ - 3’ directionality
16
Q
what are major + minor grooves
A
allows specific protein-DNA interactions, particularly in transcription
17
Q
hierarchal organisation of DNA
A
3d structure
nucleosomes
chromatin
chromatin loops
topologically associated domains
compartments
chromosome terrorties
18
Q
what is a nucleosome
A
made up of eight
histone proteins 2X( H2A, H2B, H3, H4) on
which 146 nucleotides are wound up
twice.
Linker DNA connects nucleosomes
into a “beads” structure compacting about 5 fold
19
Q
what is chromatin
A
complex of DNA and proteins (mainly histones) found in the nucleus
basic unit is nucleosome
20
Q
Chromatin Loops
A
Structures where 30 nm chromatin fibers form loops of 40-100 kb anchored to the nuclear matrix or scaffold. These loops bring distant regulatory elements like enhancers into proximity with promoters, facilitating gene regulation.
21
Q
Topologically Associated Domains (TADs)
A
: Self-interacting chromatin regions where DNA sequences within the domain interact more frequently with each other than with sequences in other domains.
TAD boundaries are marked by CTCF and cohesin and help organize gene regulation.
22
Q
A/B Compartments
A
Large-scale functional segregation of the genome into two compartments: A compartments (gene-rich, transcriptionally active, euchromatin) and B compartments (gene-poor, transcriptionally inactive, heterochromatin).
23
Q
Chromosome Territories
A
Distinct, non-overlapping regions occupied by individual chromosomes within the nucleus during interphase. Active regions are located toward the nuclear interior, while inactive regions are positioned near the nuclear periphery.
24
Nucleoside
consisting of a nitrogenous base (purine or pyrimidine) covalently bonded to a pentose sugar (ribose in RNA or deoxyribose in DNA).
Example: Adenine + Ribose = Adenosine
25
Difference Between Nucleoside and Nucleotide
Nucleoside: Base + Sugar (no phosphate).
Nucleotide: Base + Sugar + Phosphate group(s).
26
3 steps of semi conservative DNA replication
Initiation
elongation
termination
27
describe initiation
28
describe elongation + termination
29
what direction is semi conservative replication
bidirectional
30
what is in the replisome
DNA helicase
SSBs (RPA)
topoisomerase
Pol alpha (DNA polymerase alpha / DNA primase)
DNA polymerase
PCNA
RFC
FEN1 / RNase
DNA ligase
31
what are SSB's
single-stranded specific binding protein - prevents 2 strands from re-annealing
32
what is topoisomerase
reduces torsional strain on DNA caused by unwinding
33
what is PCNA and RFC
PCNA - DNA clamp that tethers DNA polymerase to DNA
RFC - auxillary factors that loads PCNA onto DNA
34
what are telomeres
non coding regions at the end of the chromosomes that prevent shortening of chromosomes during proliferation + differentiate the ends of chromosomes from DNA double strand breaks preventing end to end fusion of chromosomes
35
how does telomerase work
36
telomerase in cancer
cancer cells can keep it active
allows cancer cells to replicate infinitely without reaching replicative senescence
37
accuracy of replication
polymerases have proof reading ability that require 3' to 5' exoribonuclease activity
expand on this in detail - on spec
38
DNA repair enzymes + their importance + cancer
39
Control of Gene Expression at the Level of Transcription
promoters
transcription factors
silencers
enhancers
insulators
40
Eukaryotic Promoter Structure
Promoters are DNA sequences that initiate transcription.
Include core promoter elements, proximal promoter elements and are surrounded by distal regulatory elements
41
Core Promoter Elements
Key elements include:
TATA Box
Initiator Element (Inr)
BRE (TFIIB Recognition Element)
These elements help in RNA polymerase II recruitment and transcription initiation.
42
TATA Box
A DNA sequence (TATAAA) found approximately 25-30 bp upstream of the TSS.
It binds the TATA-binding protein (TBP), a subunit of Transcription Factor IID (TFIID).
Facilitates the assembly of the transcription initiation complex.
43
Initiator Element (Inr)
A DNA sequence centered around the transcription start site (TSS).
It helps position RNA polymerase II at the correct site for transcription initiation.
Often works together with the TATA box but can function independently as well.
44
BRE (TFIIB Recognition Element)
Located upstream of the TATA box.
Recognized by Transcription Factor IIB (TFIIB).
Helps stabilize the binding of RNA polymerase II and general transcription factors.
45
Proximal Promoter Elements
DNA sequences ~50-200 bp upstream of the TSS.
Include elements such as the CAAT box and GC box.
Bind transcription factors that recruit RNA polymerase II and other transcription machinery components.
46
Enhancer Binding Sites
DNA sequences located far upstream, downstream, or even within introns.
Bind activator proteins, increasing transcription efficiency by interacting with the promoter via chromatin looping.
47
Silencer Binding Sites
DNA regions where repressor proteins bind, inhibiting transcription.
Prevent RNA polymerase II recruitment or disrupt enhancer-promoter interactions.
48
insulator
regulate expression between neighborung genes
49
locus control region
regulates expression of a cluster of genes to ensuring correct sequence of expression
50
General Features of Eukaryotic Transcription Factors
Proteins that regulate gene expression by binding to DNA sequences near promoters, enhancers, or silencers.
Composed of DNA-binding domains and activation/repression domains.
Activator TFs: Enhance transcription by interacting with RNA Polymerase II and general transcription factors.
Repressor TFs: Inhibit transcription by blocking RNA Polymerase II recruitment or altering chromatin structure.
51
DNA-Binding Domains
Specialized regions of TFs that bind specific DNA sequences.
Examples:
Helix-Turn-Helix: Found in homeodomain proteins.
Zinc Finger: Binds DNA through zinc ions.
Leucine Zipper: Facilitates dimerization and DNA binding.
52
Categories of Transcription Factors
General Transcription Factors (GTFs): Essential for RNA Polymerase II initiation, e.g., TFIID, TFIIB.
Specific Transcription Factors: Bind to enhancers and silencers, regulate gene-specific transcription.
53
Coactivators and Corepressors
Coactivators: Proteins that interact with activator TFs and RNA Polymerase II to enhance transcription.
Corepressors: Proteins that bind to repressor TFs, preventing RNA Polymerase recruitment and altering chromatin.
54
Epigenetic Modifications
Epigenetic changes alter gene activity without changing the DNA sequence.
Include DNA methylation and histone modifications
55
DNA Methylation
Involves the addition of a methyl group (CH₃) to the 5th carbon of cytosine residues.
Typically occurs in CpG dinucleotides, often near gene promoters.
Methylation represses transcription by:
Preventing transcription factor binding
Recruiting methyl-CpG-binding proteins and histone deacetylases.
56
Effects of DNA Methylation on Gene Expression
Hypermethylation: Often leads to gene silencing (e.g., tumor suppressor genes).
Hypomethylation: Can result in gene activation or genomic instability.
Affects cell differentiation, development, and disease states like cancer.
57
Histone Tail Modifications
Histone proteins have amino acid tails on the N - terminals that can be chemically modified.
acetylation, methylation, phosphorylation, and ubiquitination.
58
Histone Acetylation
The addition of an acetyl group (CH₃CO) to lysine residues on histone tails.
Carried out by histone acetyltransferases (HATs).
Acetylation neutralizes lysine charge, resulting in a looser chromatin structure, which enhances transcription by increasing accessibility.
59
Histone Deacetylation
The removal of an acetyl group by histone deacetylases (HDACs).
Leads to tighter chromatin packing, making DNA less accessible for transcription.
Suppresses gene expression by preventing transcription factor binding.
60
Histone Methylation
addition of methyl groups (CH₃) to lysine or arginine residues on histone tails.
Can be activating or repressive, depending on which lysine residue is methylated.
H3K4 methylation: Often activates transcription.
H3K27 methylation: Typically represses transcription.
61
Chromatin Organization and Gene Activity
Euchromatin: Loosely packed chromatin, associated with active transcription.
Heterochromatin: Tightly packed chromatin, associated with gene silencing.
62
Facultative vs. Constitutive Chromatin
Facultative Chromatin: Condensed in some cell types but loosely packed in others, gene expression is cell-type specific.
Constitutive Chromatin: Always tightly packed, often gene-silent, found in areas with highly repetitive DNA (e.g., centromeres).
63
3 eukaryotic RNA polymerases and their products
RNA polymerase I -> transcribes rRNAs
RNA polymerase II -> transcribes mRNA, snRNA, snoRNA, miRNA
RNA polymerase III -> tRNA, some snRNAs
64
- mRNA
messenger, contains codons that are complementary to the anticodons on tRNA- transfers genetic information outside the nucleus
65
- rRNA
ribozyme – constituent of ribosomes, carries out protein synthesis. 28s, 18s, 5.8s made by RNA polymerase I (in nucleolus) – 5s and 7s made by RNA polymerase III (in nucleoplasm)
66
- tRNA
each RNA is specific for a particular group of amino acids – contains an anticodon complementary to a codon on mRNA, so that amino acids are sequenced in the correct positions – also has an amino acid binding site so carries the amino acid to the ribosome
67
- snRNA
small nuclear RNA – synthesised mainly by RNA polymerase II – codes for proteins of spliceosome and telomerase enzyme
68
- miRNA
micro-RNA, functions in RNA silencing and post-translational regulation of gene expression, function via base pairing with complementary sequences within mRNA molecules
69
- snoRNA
small nucleolar RNAs, guide chemical modifications of other RNAs (mainly rRNA, tRNA and snRNA)
70
RNA bases
- A, U, C, G -> uracil has replaced thymine
- Uracil is energetically less expensive to produce than thymine so it’s used in RNA
71
What is the structure of RNA Polymerase II in eukaryotes?
RNA Polymerase II is composed of 12 subunits (Rpb1–Rpb12).
Rpb1 contains the Carboxy-Terminal Domain (CTD), crucial for mRNA processing and transcription regulation.
Other subunits (Rpb2, Rpb3, Rpb5) contribute to enzyme stability and transcriptional function.
72
What is the role of the CTD (Carboxy-Terminal Domain) of Rpb1 in RNA Polymerase II?
contains repeated heptapeptide sequences (YSPTSPS).
acts as a dynamic scaffold for RNA processing factors during transcription.
CTD coordinates mRNA capping, splicing, and polyadenylation by recruiting specific proteins at different stages of transcription.
integrates transcription with RNA maturation processes, ensuring that the RNA transcript is correctly processed, modified, and stabilized before leaving the nucleus.
CTD also facilitates transition from transcription initiation to elongation, making it crucial for gene expression regulation.
73
- WHY CONVERT TO RNA
If you use DNA you are restricted to only producing 1 protein at a time
Use mRNA -> can make multiple copies which can all be translated at the same time
74
RNA structure differs from DNA structure
Single stranded
Uracil instead of thymine
Sugar backbone is deoxyribose instead of ribose
OH group on second carbon – makes RNA more reactive (emphasis in splicing)
75
RNA polymerase vs DNA polymerase
Don’t require function of a primer to synthesise a new strand = just need a DNA template and free nucleotides
Like DNA pol, they polymerise nucleotides in a 5’ – 3’ direction (this is common to all polymerases)
Orientation of gene makes a difference as to what is used as the template strand
An RNA polymerase that moves from left –> right makes RNA by using bottom strand as a template and vice versa
Non-coding strand used as a template
76
transcription - initation
77
draw out diagram for assembly of the initiation complex and describe
78
transciption - elongation
79
why does RNA have lower fidelity then DNA and does it matter
RNA polymerases do not have any proof-reading mechanisms
less important as RNA isn’t inherited
Many copies of RNA are made when a gene is expressed so a few mistakes are unlikely to affect overall levels of protein synthesis
80
transciption -Promoter proximal pause
After around 40 nucleotides have been transcribed, polymerase stops -> promoter proximal pause
From the pause state, RNA polymerase has to be released again by specific enzyme action -> addition of an additional kinase CDK9; resides in transcription elongation factor called pTEFb
Can now phosphorylate serine-2 of heptad repeat -> changes RNA polymerase so it can elongate and transcribe the rest of the gene until it reaches the terminator region
81
function of promoter proximal pause
pause allows for fine-tuned gene expression control, ensuring transcriptional accuracy and coordination
82
termination - transcription
- SIGNAL FOR TERMINATION LIES IN NEWLY FORMED RNA NOT IN DNA TEMPLATE
83
- Proteins that assist termination transcription
Rho protein -> binds to newly made RNA at C-rich G-poor regions -> scans along the RNA toward RNA polymerase in an ATP-dependant manner
When it catches up with RNA polymerase, it breaks the DNA/ RNA association and terminates transcription
