Chapter 6: The control of gene expression Flashcards
1
Q
What molecules are ribonucleotide precursors?
A
Ribonucleoside triphosphates (ATP, GTP, CTP, and UTP)
-They have three linked phosphate groups, with two being removed upon polymerisation. The energy used from this bond break is utilised to form the phosphodiester bond between the ribonucleotides.
2
Q
What bonds are formed between ribonucleotides of RNA?
A
Phosphodiester bonds
3
Q
Out of DNA polymerase and RNA polymerase, which requires a 3’ -OH group to initiate it’s function?
A
DNA polymerase needs a 3’ -OH group to initiate DNA synthesis. It is usually provided by a primer.
4
Q
In transcription, the template strand that acts as a template for the synthesis of an mRNA molecule is also called the ____________ strand and the non-template strand is also known as the _____________ strand.
A
- antisense
2.sense
(The non template strand is the sense strand because it’s sequence matches that of the mRNA that is synthesised from the template strand)
5
Q
In DNA, which groups of the nucleotides are situated at the 5’ end of the strand and at the 3’ end?
A
5’ - phosphate group
3’ - No group, 3’ carbon of the carbon ring
6
Q
On what carbon of a nucleotide is the nitrogenous base attached?
A
1’ Carbon
7
Q
What are coding RNAs?
A
mRNAs
Transcripts of protein coding genes
8
Q
In eukaryotic cells how long do coding RNAs/mRNAS usually take to fully degrade?
A
A few hours.
9
Q
What kind of RNA is rRNA and tRNA?
A
Non-coding RNAs / ncRNAs
10
Q
What are non-coding RNAs?
A
Products of non-protein coding genes
11
Q
What causes tRNA to have a specific shape?
A
The internal complementary base pairing
12
Q
in tRNA which end has a sequence exposed that can bind to an amino acid?
A
3’
13
Q
What two structural features does tRNA have that make it suitable for its function?
A
- An exposed sequence at the 3’ end that can bind to amino acids
- Three exposed bases that form the anticodon which can interact with the codon of mRNA through complementary base pairing
14
Q
What is wobble base pairing?
A
A non-Watson-Crick base pair that can occur due to the 5’ base of the anticodon having less strict pairing rules than the other 2.
15
Q
Give one example of a modified/unusual base that tRNA may contain that may be prone to wobble?
A
Inosine
16
Q
What are the four main wobble base pairs?
A
G - U
I - U
I - A
I - C
17
Q
Name the three stages of translation
A
Initiation
Elongation
Termination
18
Q
What occurs during the initiation stage of translation?
A
- Ribosomes assemble on mRNA at start of protein-coding sequence
- Initiator tRNA binds to mRNA at start codon
19
Q
What occurs during the elongation stage of translation?
A
- Second tRNA binds.
- Ribosome forms polypeptide bond between the amino acids.
- First tRNA is released
- mRNA moved through ribosome to present next codon.
- And so on…
20
Q
During polymerisation of polypeptides, to which end of the polypeptide are new amino acids added?
A
The C-terminus / Carboxyl group
21
Q
Polypeptides are always synthesised from __-terminus to __-terminus
A
N-terminus to C-terminus
22
Q
What occurs during the termination stage of translation?
A
- A stop codon is reached
- Protein synthesis stops
- Completeted polypeptides dossociates from mRNA
23
Q
Give two examples of post-translational modifications that occurs in eukaryotes?
A
- The cleaving of a polypeptide into smaller units
- Attachment of chemical groups
24
Q
Where are many of the post-translational modifications encoded?
A
Within the amino acid sequence of the polypeptide itself
25
Where does transcription happen in eukaryotes and prokaryotes?
Eukaryotes: Within the nucleus
Prokaryotes: In the cytoplasm
26
Why can translation begin whilst transcription is still ongoing within a prokaryote?
Because they both occur in the cytoplasm, unlike in eukaryotic cells which have to transport the mRNA out of the nucleus to the ribosomes, in prokaryotes transcription and translation both occur in the cytoplasm where the genetic material is free floating.
27
Name 3 post-transcriptional mRNA modifications found in eukaryotes?
- 5' Capping
- 3' Polyadenylation
- Splicing
28
What do mRNs travel through to leave the nucleus after transcription?
Nuclear pores of the nuclear envelope
29
What are the subunits of bacterial RNA polymerase?
* Four catalytic subunits: β, β’, two α’s
* One detachable regulatory subunit: the sigma (σ) factor
30
What are consensus sequences?
Highly conserved DNA sequences found within prokaryote gene promoters that are recognised by the sigma factor of the RNA polymerase
31
What contributes to the core promoter of a prokaryotic gene?
RNA binding region including:
-10 region (pinbrow)
-35 region
Transcription start site
32
What determines rate of a specific genes transcription in a prokaryotic cell?
The specific promoter sequence and whether it is 'weak' or 'strong', which determines how often RNA polymerase will bind
33
What number is used to mean 'transcription start site'?
+1
34
What occurs to change the closed promoter complex that formed upon the binding of RNA pol and the sigma factor, into an open promoter complex?
The unwinding of DNA strands which form a transcription bubble.
35
How soon can a new RNA polymerase (holoenzyme) attach after transcription in prokaryotes?
As soon as the promoter has been cleared by the first RNA polymerase, which signals the end of initiation, another RNA polymerase is capable of binding.
36
What are the two types of transcription factors in prokaryotes?
- transcriptional activators: promote the recruitment of RNA Pol to the promoter
-transcriptional repressors: repress the recruitment of RNA Pol to the promoter
37
Describe the two types of domains of transcription factors?
- DNA binding domains: interact with backbone and bases of DNA
-Transactivation domains: Interact with other transcription factors or proteins bound to the core promoter
38
What is a DNA binding motif?
Common structures characteristic of DNA binding proteins which are specialised in a way that it has binding specificity with a specific DNA sequence.
39
Describe the structure of the bacterial lac operon (draw it maybe)
From 3' - 5'
Lac control region:
- Activator site (CAP site)
- Promoter region
- Operator
Three genes with related function of metabolising lactose:
- lacZ
- lacY
- lacA
40
What kind of transcript is produced from the transcription of the bacterial lac operon?
A polycistronic mRNA transcript
41
What is allosteric regulation?
Regulation of protein activity by the binding of an effector molecule at a site other than the active binding site.
42
What happens to the lac operon when lactose is not present?
lac repressor binds to the operator and prevents RNA Pol Holo binding, therefore preventing transcription
43
What happens to the lac operon when lactose is present?
Allolactose (a metabolite of lactose) acts as an effector molecule and binds to the repressor causing it to separate from the operator, therefore allowing RNA Pol Hol to bind and transcribe.
44
What happens to the lac operon when there is low glucose?
signalling molecule cAMP (inversely proportional to glucose in the cell) binds to CAP causing conformational change and allowing CAP to bind to the activator site increasing transcription levels
45
What happens to the lac operon when there is high glucose?
No cAMP is made to bind to CAP and therefore CAP cannot bind to the activator site and transcription occurs but at a low level
46
What is a transcription terminator?
An RNA sequence (transcribed from the DNA template strand) that signals RNA polymerase to terminate RNA synthesis
47
What are the two types of transcription terminatorsfound in bacterial RNA?
- Rho dependent
Sequence that acts as binding site for Rho factor (protein) that unwinds RNA from DNA and inteferes with complementary base pairing.
-Rho-dependent
An inverse repeat sequence that causes RNA to fold back on intself in a hairpin loop facilitated by complementary base pairing which destabilises the interaction between RNA Pol and the DNA-RNA structure.
48
What are attenuators?
Premature termination sequences
Commoly used by bacteria to downregulate gene expression.
Sequences cause hairpin loops near the 5' of the RNA which prevents full transcription.
49
What usually signals attenuation during transcription?
Environmental signals
50
What faciliates the binding of RNA polymerase to mRNA in eukaryotic transcription?
General transcription factors
51
What are the three types of RNA polymerase in eukaryotic cells and what type or RNA are they responsible for synthesising?
RNA polymerase I - rRNA
RNA polymerase II - mRNA (also most small nuclear RNA/snRNA, and microRNA)
RNA polymerase III - tRNA (small RNAs, other small RNAs)
52
How many subunits make up RNA polymerase II? How many of these are core subunits?
10 - 12
3 core subunits
53
Where is the TATA box located in a eukaryotic gene?
Inebwteen the -35 and -25 mark
54
What transcrption factor recognises the TATA box?
TFIID (Transcription factor 2 D)
55
What is TFIID?
A protein complex formed of:
- TATA binding protein (TBP)
-TBP-associated factors (TAFs)
56
What are the two types of sites found in the proximal promoter region of a eukaryotic gene, and what binds to them?
- Inducers
- Repressors
Specific transcription factors bind
57
How far upstream is the proximal promoter region usually located?
-250bp
58
How far is the distal regulatory region usually located from the transcription start site?
50kbp+ upstream or downstream
59
What two types of sequences are found in the distal regulatory regtion and what binds to each of them?
- Silencers (bounded to by transcriptional repressor proteins)
- Enhancers (bounded to by transcriptional activator proteins)
60
How do transcription factors that bind to the distal regulatory region allow interaction between that region and the promoter region?
Specific transcription factors bind to silencer or enhancers and cause conformational change of the DNA, meaning it can fold and end up a lot closer to the promoter
61
Name 3 differences between euchromatin and heterochromatin.
1. Heterochromatin is densely packed, euchromatin is loosely packed
2. Heterochromatin has few genes, euchromatin has many genes
3. Heterochromatin is trancriptionally inactive, euchromatin is transcriptionally active
62
Describe the process that makes previously inaccessible DNA accessible by modification of chromatin structure.
1. Transcriptional activators bind to histones and attract histon acetylases
2. Hyston acetylases catalyse addition of acetyl grouo to lysine within the histone
3. Chromatin remodelling proteins are attracted and remove or slide nucleosomes
4. Transcription factors and RNA Pol. can now bind
63
What are the main roles of general transcriptio factors? (3)
1. Correctly position RNA polymerase at promoter
2. Separate DNA strands
3. Release RNA Pol from transcription initiation complex to it can transcribe
64
What forms the Transcription initiation complex?
RNA polymerase and general transcription factors
65
What is combinational control?
The eukaryotic transcription factors working as an organised group to determine the expression of a gene.
66
What is the mediator complex?
A protein complex (20+) that acts as an intermediary between RNA Pol and general transcription factors bound to the core promoter and specific transcription factors bound to the proximal promoter or more distant regulatory regions.
67
What are the steps of transcription initiation in eukaryotes?
1. TFIID recognises TATA box
2. General transcription factors recruited to faciliate binding of RNA Pol
3. RNA polymerase binds
4. RNA pol separates DNA strands and forms transcription bubble
5. Phosphorylation of RNA pol causes conformation change causing it to be released from transcription inititation complex, begins to move down DNA
6.... elongation
68
Desribe how you could study the regulatory potential of sequences of a gene promoter region?
1. Isolate regulatory sequence to be studies
2. Insert regulatory region into a DNA plasmic upstream of reporter gene
3. Introduce plasmic DNA into cells
4. Measure reporter protein
(Good example to use is GFP that causes green fluoresent light)
69
What is the function of elongation factor proteins in eukaryotes?
To reorganise nucleosomes to allow RNA polymerase to progress along the DNA strand without dissociating.
70
What gene regulation mechanism can occur in transcription termination?
Premature termination
71
What is the function of the 5' cap of mRNA?
- Enables transportation from nucleus through nuclear pores
- Essential for ribosome binding and translation
- Prevent degradation by cytoplasmic enzymes
72
In 5' capping, what molecule is added to the 5' end of mRNA and which enzyme catalyses this reaction?
- Guanosine triphosphate (GTP) nucleotide
- Catalysed by Guanylyl transferase (part of RNA Pol II complex)
73
What is cap-snatching?
When a cell does not cap its own genome but instead steals pre-formed caps from it's hosts mRNA, also preventing hosts mRNA being translated e.g., influenza
74
What is added to the 3' end of an mRNA transcript in eukaryotic cells?
Approx. 200 adenosine nucleotides that form a Poly(A) tail
75
What is the function of the poly(A) tail that is added to mRNA after transcription?
- Binding site for poly(A) binding protein which:
1. Prevents degradation
2. PRomotes export from the nucleus
3. promotes translation
76
What is mRNA splicing?
The removal of introns from the primary transcript, and the joining together of the remaining exons.
77
What is alternative splicing? What does this result in?
When different combinations of exons are encorporated into a mature mRNA during splicing, forming splice variants that result in different proteins being made in different cells and tissues, despite using the exact same gene.
78
What are ribonucleases?
Enzymes that degrade the poly(A) tail of mRNA, shortening it over time.
79
The amount of mRNA molecules in a cell reflects what? (2)
- How fast the mRNA is being made
- How fast the mRNA is being broken down
80
Give an example on how proteins can regulate their own level of mRNA?
Tubulin monomers, when plentiful within a cell, can bind to tubulin mRNA subsequently making it more succeptible to breakdown by ribonucleases, therefore preventing the synthesis of more tubulin.
81
What type of RNAs are microRNAs?
Small ncRNAs (nuclear RNAs)
82
What do MicroRNAs do in terms of gene regulation?
Target specific mRNAs for degradation or inhibit their translation
83
What is translation?
The process of synthesising a polypeptide that is carried out my a ribosome using the information encoded in the mRNA sequence to determine the amino acid sequence of said polypeptide chain.
84
Name the subunits of prokaryote and eukaryotic ribosomes/
Prokaryotes: 70S ribosomes
- Small (30S) subunit
- Large (50S) subunit
Eukaryotes: 80S ribosomes
- Small (40S) subunit
- Large (60S) subunit
85
What are the three stages of translation?
1. Initiation
2. Elongation
3. Termination
86
Outline the steps of translational initiation in prokaryotes.
1. 30S subunit binds to ribosome binding site, facilitated by translation initation factor proteins (IF1 & IF2) which also prevent 50S subunit from binding
2. Initiator tRNA with (IF2 and bound GTP) carries methionine to small subunit
3. Hydrolysis of GTP to GDP + Pi releases energy which is used to attach 50S subunit to 30S subunit
4. IF1, IF2, and IF3 dissociate from ribosome complex
5. Translation initiation is complete
87
Outline the steps of translational initiation in eukaryotes.
1. Ribosome complex forms of small 40S ribosome subunit, initiatiator tRNA, and a translation initiation factor (eIF2) that is bound to GTP
2. Complex attaches on 5' cap, faciliated by eIF3 and eIF4
3. Complex scans along mRNA
4. First translation initiation codon, recognised by the consensus/kozak sequence '5' ACCAUGG 3', is located
5. GTP is hydrolysed, energy released is used to attach large (60S) subunit
6. Initiation factors disociate
7. Initiation is complete
88
What are the main translation initiation factors found in prokaryotes?
IF1, IF2, and IF3
89
What are the main translation initiation factors found in eukaryotes?
eIF2, eIF3, and eIF4
90
What is the biggest difference between translation initiation in prokaryotes and eukaryotes?
Prokaryotes have a ribosome binding site on the mRNA
or
The formation of a initiation complex in eukaryotes
91
What are the names for the two sites found in ribosomes?
P (polypeptide) site
A (acceptor site) site
92
What are polyribosomes/polysomes and where are they found?
Clusters of ribosomes all bound to a single mRNA molecule within the cytosol
93
What are the steps of translational elongation?
1. Transcriptional elongation factor with bound GTP brings apprpriate tRNA to A site
2. Elongation factor hydrolyses GTP to GDP and inorganic phosphate causing conformation change of ribosome that allows the tRNA to enter A site
3. Ribosome catalyses formation of peptide bond between amino acids in P and A site
4. Elongation factor hydrolyses carries GTP molecule causing conformational change of ribosome that triggers translocation
5. mRNA moves along three nucleotides so tRNA in A site moves to P site, the next codon enters A site, and the new tRNA occupies it
6. Amino acid bound to tRNA that was first in P site is released
7. Said tRNA leaves ribosome
8. Repeat
94
In which direction does polypeptide synthesis occur?
From N-terminus to C-terminus
95
Which elongation factors are involved in prokaryotic translational elongation?
EF-Tu and EF-G
96
Which elongation factors are involved in eukaryotic translational elongation?
eEF1 and e-EF2
97
What is translocation in regards to translation?
The process that advances the mRNA-tRNA along the ribosome to allow the next codon to enter the A site to be decoded
98
What needs to be detected for translation to be terminated?
Termination codon
99
What happens in translation once a termination codon has been reached
- Release factor (RF) enters A site that recognises one of three termination codons (UGA, UAG, UAA) which ends elongation.
- Polypeptide is released from the tRNA in P site
- Ribosomal subunits released back into cytoplasm
100
Name 2 mechanisms that regulate the initiation of translation?
- Secondary structure in mRNA inhibiting recruitment of small ribosomal subunit
- RNA binding proteins recruit small ribosomal subunit
101
Which protein complex degrades incomplete or misfolded proteins?
Proteasome
102
What is the half-life of a protein very dependent on?
N-terminus amino acid - regions rich in Pro, Glu, Ser and Thr (PEST sequences) mark proteins for rapid degradation
103
Name 5 post-translational modifications?
Additional folding
Binding to other polypeptides
Enzymatic cleavage
Phosphorylation
Glycosylation
