Block A Flashcards
Where does translation take place?
Cytosol
What is a nucleoside?
A base joined to a sugar
What is a nucleotide?
A nucleoside joined to one or more phosphate group
What is a phosphate group?
A phosphorus atom bonded to 4 oxygen atoms (PO₄³⁻)
Where are the sugar phosphates and bases on a DNA strand?
The sugar phosphates on outside, bases inside
Roughly how many bases are in each DNA turn?
10
What is intron short for?
intervening DNA
What is exon short for?
expressed DNA
What stage of the cell cycle is DNA replicated?
S-phase (synthesis phase)
What makes DNA replication semiconservative?
one strand of the DNA is maintained every time its duplicated (original strand is split in 2 making each half making new strand half original half new)
What direction is a DNA strand synthesised by DNA polymerase?
5’ to 3’
How does DNA polymerase synthesise DNA?
catalyses the step-by-step addition of deoxyribonucleotide units to DNA
What is needed by DNA polymerase to synthesise new DNA ?
A primer (that has a free 3’ -OH)
What is PPi?
Pyrophosphate
The base sequence of mRNA is the complement of which DNA strand?
Template strand (is a copy of coding strand but with U for T)
What are the 3 stages of mRNA synthesise, in order?
- initiation
- elongation
- termination
Where does the initiation stage of mRNA synthesis occur in the DNA?
At promoters
What is a promoter?
A defined DNA sequence near the transcription start site
Give 3 examples of promoter sequences
- TATA box
- CAAT box
- GC box
How do promoter sequences define the point of transcription?
BY recruiting RNA Polymerase II
Which strand can the CAAT and GC boxes be on?
PLEASE HELPPPPP
The template (antisense) strand and more commonly the coding (sense) strand
(Lecture 1, Slide 23)??????????
What is the initiation stage of mRNA synthesis regulated by?
transcription factors (TF)
What can sequences known as “enhancers” influence?
Gene expression
What are enhancer sequences binding sites for?
transcription factors
Are transcription factors specific?
Yes
How are transcription factors specific?
They recognise DNA sequences near promoters of genes
The template (antisense) strand and more commonly the coding (sense) strand
(Lecture 1, Slide 23)
signaling pathways
What can transcription factors be activated by?
phosphorylation and subsequent movement into the nucleus
In the elongation phase of mRNA synthesis which strand is unwound?
The coding strand
What is created during the elongation stage of mRNA synthesis
A RNA-DNA hybrid helix of mRNA and the template strand
Is mRNA stable?
No
Give 2 ways mRNA is processed to make it more stable in the termination stage of mRNA synthesis
- modified 5’ cap
- poly A tail at the 3’
Give 2 reasons the modified 5’ cap and poly A tail at the 3’ end are added in the termination stage of mRNA synthesis
Help stability and translation
What codon does protein synthesis always start with?
AUG
What amino acid does the codon AUG code for
Methionine
What is the tRNA that carries amino acids to the site of translation?
aminoacyl tRNA
How does tRNA form its clover shape?
Hydrogen bonding between bases
What amino acids attached to, and where, on a tRNA molecule?
They are attached to an aminoacyl moiety at the amino acid attachment sie
What is present at the amino acid attachment site on a tRNA molecule?
A flexible CAA arm
What is the purpose of aminoacyl tRNA synthesis
To link a specific amino acid with a specific tRNA
What decided what amino acid links with the tRNA molecule?
The anticodon
What is first stage of aminoacyl synthesis called?
The amino acid activation step
Give the equation for the first stage of aminoacyl synthesis
Amino acid + ATP —-> Aminoacyl-AMP +PPi
What is second stage of aminoacyl synthesis?
Transfer of aminoacyl-AMP to a specific tRNA
Give the equation for the second stage of aminoacyl synthesis
Aminoacyl-AMP + tRNA —-> aminoacyl-tRNA + AMP
What is the hydrolysis reaction that drives aminoacyl tRNA synthesis
The hydrolysis of pyrophosphate (PPi)
Give the equation for the hydrolysis of PPi
PPi + H2O —-> 2Pi
Give the full equation for aminoacyl tRNA synthesis
Amino acid + ATP + tRNA + H20 —-> Aminoacyl-tRNA + AMP + 2Pi
Where does the attachment of amino acids to tRNA occur?
At an activator site of aminoacyl tRNA synthetase
What is the proof reading ability of aminoacyl tRNA synthetase able to do?
“Reject” incorrect amino acids
What makes aminoacyl tRNA synthetases specific?
They recognise the tRNA structure and anticodon
How do aminoacyl tRNA synthetases proof read?
The CCA arm can move amino acids between the activation and editing sites
If the amino acid fits well into the editing site, it is removed by hydrolysis
What are the subunits of ribosomes made up of?
Ribosomes are made up of 2 rRNA subunits
Give the function of the “P” site of a ribosome
Where the peptide grows (through a “tunnel” in structure)
Give the function of the “A” site of a ribosome
Aminoacyl-tRNA binds here, bringing the next amino acid
Give the function of the “E” site of a ribosome
Exit site for empty tRNAs
Which special tRNA bears methionine in order to help initiate translation?
Met-tRNAi (i is a subscript)
Give 3 things Met-tRNAi forms a complex with in order to help initiate translation
- initiation factors
- GTP
- 30s ribosomal subunit
Give 2 things Met-tRNAi, initiation factors, GTP and 30s ribosomal subunit recruit once they find an AUG codon
What complex does this form?
- recruits remaining 50s subunit
- recruits further initiation factors
Forms the 80s initiation complex
What does the formation of the 80s initiation complex result in?
protein synthesis occuring
Give the 3 regulatory steps of transcription/translation
- selective control of gene expression
- control of mRNA synthesis rate/stability/processing
- protein synthesis/degradation rates
Which enzyme synthesises the primary transcript of mRNA before it is spliced?
RNA polymerase
When does mRNa capping occur?
When the pre-mRNA is ~ 25nt long
What is required to cap the pre-mRNA?
Specific capping proteins
What enzyme cleaves the phosphodiester bond to e when mRNA is being made?
Endonuclease (DOUBLE CHECK THIS CARD 2 slide 9)
Give the name of the specific adenine which is key to splicing every intron
The branch site
What is present as the mRNA is made?
An AAUAAA cleavage signal
What do all intron end with?
AG
Endonuclease cleaving the phosphodiester bond when mRNA is being made allows what?
Poly(A) polymerase to function
What does poly(A) polymerase do after it has been activated by endonuclease cleaving the phosphodiester bond when mRNA is being made?
Adds a poly(A) tail to the pre-mRNA
What gives a poly(A) tail its name?
It is a polymer tail of multiple adenine nucleotides (A)n where n can be >250
What do splicing proteins recruit and what complex does this create?
Other proteins to form complex called spliceosome
What do all introns begin with?
GU
Give 2 thins present in every intron which are key to splicing
- pyrimidine rich tract (Py)
- specific adenine
Give 3 things the spliceosome can do to RNA and in what way
Capture, splice and release RNA accurately and in a coordinated way
Give the first step to RNA splicing
Splicing proteins recognise 2 specific sites, the GU at the 5’ splice site and the branch site
What does the spliceosome capturing, splicing and releasing RNA require?
Careful choreography of spliceosome components
What form are the introns released in, in RNA splicing?
lariat form
What is alternative RNA splicing?
A process where different mRNAs are generated from the same initial primary transcript
What can alternative RNA splicing result in?
Proteins with different functions being formed
Why can some RNA molecules self replicate?
They can fold into distinct structures ( having the capaity to act as an enzyme and splice themselves)
Who discovered RNA can act as an enzyme and splice itself?
- Tom Cech
- Sidney Altman
What is RNA interference (RNAi)
A powerful tool to distrupt gene expression
How was RNAi discovered?
Double stranded RNA was introduced to a cell and it suppressed the transcription of genes that contained sequences present in the original double stranded RNA
What does RNA interference (RNAi) rely on?
The key activity of small interfering RNAs (siRNAs)
How can exploitation of RNA interference be used in cell biology?
To examine the function of a particular hene product
Give 3 potential uses of CRISPR
- Create cell lines to probe specific gene function
- Produce specific mutations in the germ line
- used in ex vivo and in vivo genome editing for clinical therapy
Give 3 reasons why cells signal
- Co-ordination of day-to-day physiology
- Regulate behaviours in multicellular organisms
- Cells respond and change to stimuli in their environment
Give 5 physiological activities that cell signaling regulates
(answers include):
1. cell metabolism
2. cell growth
3. cell division
4. cell motility
5. cell morphology
6. cell death
7. Co-ordination and development of gene expression
What is intercellular signalling?
Communication between cells
What does intercellular signalling permit?
A single cell to influence the behaviour of other cells in a specific way
What is intracellular signalling?
Signalling within a single cell
What does intracellular signalling happen in response to?
extracellular or intracellular stimuli
What is autocrine intercellular signalling?
A cell targets itself
(double check this one)
What is paracrine intercellular signalling?
A cell targets a nearby cell
What is endocrine intercellular signalling?
A cell targets a distant cell through the blood stream
What is the juxtacrine signalling?
A cell targets a cell connected by gap junctions
Is cell signalling a linear process?
NO
Give the 4 major types of cell signalling receptors
- ligand-gated ion channel receptors
- G-protein coupled receptors
- kinase-linked receptors
- nuclear receptors
What can alter the “what”, “where” and “when” parameters of G-protein coupled receptor function?
Different signals
Give 3 biases that can alter the kinetics of the response of cell signalling pathways
Bias with extracellular signal, intracellular signal, or both
What are signalosomes composed of?
A unique combination of signalling pathway components
How are signalosomes targeted to discreet intracellular localisation?
via the association of anchor or adaptor proteins
How adaptable and dynamic are signalosomes?
Very
What do signalosomes allow?
Cells to construct the optimum cell subdomain for signalling
Where can G-protein coupled receptors generate unique signals from?
intracellular compartments
Where are G-protein coupled receptors internalised to?
Endosomes and then trafficked to a pre-Golgi compartment
What is the purpose of compartmentalisation of cAMP signalling?
Cells need a way to “turn off” signalling
What catalyses the breakdown of cAMP?
Phosphodiesterases
What can the compartmentalisation of cAMP create?
cAMP gradients in the cytosol
Can cells have more than one cAMP gradient?
Yes
State the 4 major types of signalling pathway receptors and the typical times associated with these
- ligand-gated ion channel receptors (milliseconds)
- G-protein coupled receptors (seconds)
- Kinase-linked receptors (hours)
- nuclear receptors (hours/days)
How can one signal generate many responses?
Different cells can have the same receptor
What makes different types of cells different?
All cells contain the same DNA sequences (genes) but are made different by which genes are expressed, this is different in different cell types
What is meant by a gene being “expressed”?
it is transcribed into mRNA leading to a production of the corresponding protein
What has a major role in determining what genes are expressed?
Epigenetics
What is epigenetics?
Modifications to the genome that affect gene expression but do not alter the DNA sequence
What are CpGs?
A rare dinucleotide sequence
What does the p in CpG islands represent?
The phosphate residue on the DNA backbone
What is methylation associated with?
Gene silencing
How does methylation cause gene silencing?
The methyl group protrudes into the major groove of DNA which interferes with the binding of transcription factors
What are CpG islands?
Regions of the genome which have a high density of CpGs
Where are CpG islands mostly found?
The promoter region of genes
How do CpG islands alter the binding of transcription factors?
By undergoing chemical modification (methylation)
What bases is methylated by CpG
cytosine
How does methylation prevent the binding of transcription factors?
Methyl binding proteins bind to the methylated DNA preventing the binding of transcription factors
What is a nucleosome?
A DNA / histone octamer complex
What is the overall DNA-histone complex referred to as?
Chromatin
How many histone subunits form a histone octamer?
4
What happens after 4 histone subunits form an octamer?
DNA wraps around the octamer
What are the 2 chromatin states?
Euchromatin and heterochromatin
Give 3 differences between euchromatin and heterochromatin
- Euchromatin is loosely packed whereas heterochromatin is tightly packed
- Euchromatin is enriched in genes whereas heterochromatin is usually genetically inactive
- Euchromatin is under active transcript whereas heterochromatin cannot be accessed by RNA polymerase
Give 2 things regulate chromatin state
Methylation and acetylation of the histones
Give 2 ways in which epigenetic changes can lead to cancer
- Phosphorylation of a tumour suppression gene could inhibit gene expression.
- Genes could be turned on by changing histone modification pattern
