3.1.3 Regulation of gene expression and 3.1.4 Transcription, RNA processing and translation Flashcards
1
Q
Where do transcription and splicing occur in eukaryotes?
A
In the nucleus (compartmentalisation)
2
Q
Where does translation occur in eukaryotes?
A
In the cytoplasm (compartmentalisation)
3
Q
Do prokaryotes show compartmentalisation during gene expression?
A
No, all three processes occur in the cytoplasm (no membrane-bound organelles).
4
Q
What proteins carry out transcription?
A
RNA polymerases (four types in eukaryotes, one type in prokaryotes)
5
Q
What organelle is involved in translation?
A
Ribosomes
6
Q
What monomer is used to form RNA?
A
Ribonucleoside triphosphates
7
Q
What do RNA polymerase enzymes use to form the RNA transcript?
A
They use one of the strands of DNA as a template (the strands are separated by a helicase component of the enzyme itself), and the other strand is known as the ‘coding strand’ as the transcript will have a similar structure (thymine, T, bases replaced by uracil, U, bases) to that strand.
8
Q
In which direction do RNA polymerases form transcripts?
A
5’ to 3’ (so add on to the 3’ end), same as DNA polymerases - therefore moves along the template strand in a 3’ to 5’ direction. Either strand can be transcribed, depending on the direction/orientation of the polymerase enzyme.
9
Q
What is the main difference between DNA and RNA polymerase enzymes?
A
DNA polymerases require a primer, RNA polymerases do not.
10
Q
What are the products of RNA polymerase I?
A
rRNAs (ribosomal RNAs)
11
Q
What are the products of RNA polymerase II?
A
mRNAs (messenger RNAs), snRNAs (small nuclear RNAs), snoRNAs (small nucleolar RNAs) and miRNAs (micro RNAs)
12
Q
What are the products of RNA polymerase III?
A
tRNAs (transfer RNAs) and some snRNAs (small nuclear RNAs)
13
Q
What are the products of RNA polymerase IV/POLMRT?
A
Mitochondrial RNA polymerases transcribe mtRNA - acts upon mitochondrial DNA and transcribes the products there.
14
Q
What is the function of mRNA?
A
To act as a messenger and code for proteins
15
Q
What is the function of rRNA?
A
To form the core of ribosomes and catalyse protein synthesis
16
Q
What is the function of tRNA?
A
To serve as an adaptor between mRNA and amino acids during synthesis - is bound by an ester bond to an amino acid molecule.
17
Q
What is the function of aminoacyl-tRNA synthetase/tRNA ligase?
A
This enzyme causes ATP to bind to the amino acid, losing two phosphate groups in the process to form amino acid-AMP. The enzyme then facilitates the transfer of this aminoacyl group to the tRNA molecule, as the AMP is swapped for the tRNA and an ester bond formed. This results in the amino acid to be attached to the tRNA molecule.
18
Q
What is the function of miRNAs?
A
These regulate gene expression as part of RNA interference mechanisms - after associating with a RISC factor (RNA-induced silencing complex), these small, single stranded RNA molecules are able to bind to complementary codes on mRNA molecules and occasionally, through use of the Argonaute protein, are able to cause the cleavage of the mRNA, leaving it open to degradation by exonucleases. More frequently, however, the miRNA-RISC complex just initiates the association of more of these complexes, causing translation to be prevented and the mRNA molecule to be shipped off to a protein- or P-body, where the molecule is sequestered from ribosomes and stored/eventually degraded.
19
Q
What is the function of snRNAs?
A
Primary function is pre-mRNA processing within the nucleus - they are involved in regulating splicing through binding to short conserved motifs between exon regions.
20
Q
What is the function of snoRNAs?
A
Located within the nucleolus, some are used in the processing of rRNA but most just act as guides for other cellular RNAs (mostly snRNAs).
21
Q
What are the bases available in RNA?
A
Adenine (A), Uracil (U, replaces thymine), Cytosine (C) and Guanine (G)
Complementary pairings: A U, C G
22
Q
Where do RNA polymerases associate on DNA strands to initiate transcription?
A
Gene core promoters
23
Q
What is an example of a promoter region?
A
TATA boxes are most common
24
Q
What are the molecules that facilitate the association of RNA polymerase to the promoter region?
A
Transcription factors (proteins - multiple in eukaryotes, often only one in prokaryotes)
25
Which genome is more complex, prokaryotic or eukaryotic?
Eukaryotic genomes are more complex
26
What do eukaryotic RNA polymerases require that prokaryotic RNA polymerases do not?
The presence of multiple transcription factors, i.e. GTFs (general transcription factors) that link the protein scaffold complex formed at regulatory sites to the RNA polymerase enzyme.
27
Where do transcription factors bind?
Either directly to the promoter region (GTFs only) or in other cis-regulatory (cis = on the same chromosome - and example of a trans-regulatory site would be a gene which codes for a relevant transcription factor on a different chromosome) site just upstream of the promoter region.
28
What is a repressor?
This is a transcription factor that inhibits the action of RNA polymerases, for example by completely blocking the promoter region, preventing binding.
29
What is an activator?
This is a transcription factor that encourages the action of RNA polymerases, for example by providing more regions that can form favourable bonds with the enzyme (incr stability) other than just the DNA strand, and by positioning the enzyme so that it is facing in the correct direction for transcription.
30
What is a co-repressor?
This is another name for repressors but in eukaryotic cells - as there are often multiple transcription factors needed, the nomenclature changes, and these factors do not interact directly with the enzyme.
31
What is a coactivator?
This is another name for activators but in eukaryotic cells - as there are often multiple transcription factors needed, the nomenclature changes, and these factors do not interact directly with the enzyme.
32
How do transcription factors/GTFs bind to DNA?
This is due to various regions on the proteins (i.e. hydrophobic areas, electron donating or accepting regions) that recognise specific sequences on the DNA strand - because of this, gene expression is highly regulated as each transcription factor can be specific to a promoter region and an RNA polymerase enzyme. They can also bind cooperatively, forming dimers before function is allowed, and different factors can have different regulatory effects depending on the combination they are in.
33
Give examples of GTFs/general transcription factors for RNA polymerase II.
TFIIA, TFIIB, TFIID, TFIIF, TFIIE, TFIIH
34
What is a pre-initiation complex (PIC)?
This is the scaffold of proteins formed at a promoter/regulatory region that control transcription and allow for its initiation.
35
* Go through the process of GTF association for RNA polymerase II
TBD subunit of TFIID associates with a TATA box
TFIIB joins through recognising and associating with a BRE sequence on the TFIID
TFIIF recruits RNA polymerase II and stabilises its interaction with TFIIB and TBP
TFIIE and TFIIH join (subunits of the TFIIH protein have helicase properties, which is required to unwind sections of DNA)
After this point, the pre-initiation complex has been formed
(Subunits of the TFIIH also have kinase activity which will phosphorylate the C-terminal domain of Pol II, which ends abortive initiation/triggers transcription).
36
Why are transcription factors useful?
They are able to interact with/be affected by the environment, allowing the cell to have timely and relevant responses to tissue specific and proliferation cues as well as various stimuli.
37
Which regulatory complexes are more complicated?
Eukaryotic
38
What are distal regulatory elements?
Regions of DNA that can regulate gene expression but can be up to 1mb away from the promoter region itself
39
What is DNA looping?
This is where the stands of DNA are looped through the action of proteins to bring regulatory and promoter regions physically closer together. This allows for better interaction between the two regions and proteins associated at either sequence. The method is seen in both prokaryotes and eukaryotes.
40
What is the function of an enhancer element?
To activate expression of a promoter region over long distances
41
What is the function of a silencer element?
To repress transcription at promoters
42
What is the function of an insulator element?
This is used to regulate expression between neighbouring genes/prevents other regulatory sites from affecting genes other than their target.
43
What is the function of a locus control region?
These regulate the expression of a cluster of genes to ensure correct sequence of expression.
44
What allows distant sequences to impact upon transcription?
DNA-protein and protein-protein interactions.
45
What component of eukaryotic DNA allows for more regulation than that of prokaryotes?
Chromatin structure and the presence of nucleosomes/histones.
46
How can histones affect gene expression?
Due to the opposite charges between histones and DNA (histones contain many basic amino acids, so are positively charged, whereas the backbone of DNA contains many phosphate groups so has a high negative charge), the two have high electrostatic attraction and so associate quite closely.
This can be altered through histone modifications, however.
DNA must actually be accessible for the association of transcription factors to occur - chromatin and histone modifications can affect this.
47
What is the term for dense chromatin (many nucleosomes)?
Heterochromatin - also associated with additional heterochromatin proteins that bind to the specifically modified histone tails in that region
48
Genes in heterochromatin are...
Effectively silenced, gene expression is prevented.
49
What is the term for 'looser' chromatin (fewer nucleosomes)?
Euchromatin - can be regulated by histone tail modifications. Histones are positioned further apart.
50
Genes in euchromatin are...
Able to be transcribed - these regions often contain active genes.
51
What are the general features of eukaryotic transcription factors?
DNA binding regions
Regions to associate with other regulatory proteins or associate directly with the polymerase enzyme itself (latter is GTFs only)
52
What are potential histone tail modifications?
Acetylation (addition of an acetyl group, CH3CO)
Methylation (addition of a methyl group, CH3)
Phosphorylation (addition of a phosphate, PO4^3-)
53
Which terminal do histone tails express?
N-terminal
54
What can be the effect of histone tail modifications?
A gene can either be silenced (due to the promoter region becoming closely bound to the histone, preventing transcription factors association) or activated (DNA unwound from histone due to modification, allows for binding of transcription factors and initiation of transcription).
55
What is the affect of acetylation of histones?
By adding negatively charged acetyl groups, DNA dissociates from the histone protein due it being repelled by the like charge/the basic charge of the histones is reduced.
The opposite occurs/DNA associates more closely during deacetylation.
56
What enzymes carry out acetylation and deacetylation of histones?
HATs carry out acetylation (histone acetyl transferases)
| HDACs carry out deacetylation (histone deacetylases)
57
What is the effect of methylation of histones?
DNA is activated/dissociates from the histones after methyl groups are added, due to the CH3s binding to the basic amino acids that make up the histone protein - this reduces the positive charge of the histone and causes the electrostatic attraction between the two species to be decreased.
The opposite occurs/DNA associates more closely during demethylation.
58
What enzymes carry out methylation and demethylation of histones?
Lysine- and arginine-specific methyltransferases carry out methylation
Demethylase enzymes remove methyl groups from nucleic acids (especially histones)
59
What is the effect of methylation of DNA and where does it occur?
This occurs most commonly at CpG sites (regions where cytosine and guanine bases are adjacent) and methylation of DNA prevents the action of RNA polymerase enzymes, so silences genes/prevents transcription.
The opposite occurs/transcription is possible once the methyl groups are removed.
60
What enzyme maintains methylation of DNA?
This is the Dnmt1 enzyme (DNA methyltransferase 1 enzyme)
61
What is the effect of phosphorylating histones?
Phosphate groups are highly negative, so will cause dissociation of DNA, but phosphorylation of histones also allows for histone sliding, where the histones - as they are dynamic structures - are able to 'slide' up and down the DNA strand, so will therefore affect different regions of DNA.
62
What enzymes carry out the phosphorylation and de-phosphorylation of histones?
Kinases phosphorylate, phosphatase enzymes remove phosphate groups.
63
What regions of DNA are likely to be heterochromatic?
Regions of DNA that don't contain any genes, or that contain inactivated that are not necessary within the cell.
64
* What is an operon?
This is a series of genes in sequence (more commonly seen in prokaryotes) that all code for the same enzyme.
65
* How did the study of operons lead to the Jacob-Monod model of gene regulation?
Through observations of the trp and lac operons in bacteria (E.coli), the latter in particular. The Jacob-Monod model suggested that a region of DNA just upstream of the transcription initiation site (promoter region) has the ability to associate with transcription regulators/factors.
In the trp model, the enzyme coded by the operon catalyses the formation of the amino acid tryptophan -however, when the bacteria is in a solution with a high conc of the amino acid, this operon is repressed/the production of the enzyme halted. This is because the amino acid binds to an inactivated repressor protein in the cytoplasm and activates it (x4 molecules needed), meaning it can then bind to its complementary regulatory site and inhibit the action of the prokaryotic RNA polymerase enzyme. Once the extracellular conc of tryptophan decreases, the factor dissociates and action of RNA polymerase is allowed again.
In the lac model, the enzyme coded for by the operon catalyses the digestion of lactose sugars. When the enzyme is in a solution with a high conc of glucose, this enzyme is not needed and so is repressed by the lac repressor protein. The lac repressor protein is an indirect sensor for lactose, becoming inactivated when its isomer, allolactose, detects the disaccharide. Catabolite activator protein (CAP) acts as an activator protein when the concentration of lactose is high and glucose is very low - this protein is able to indirectly sense when the glucose concentrations are low (through cAMP messengers), only then activating the lac operon at an upstream regulatory site.
66
What is constitutive heterochromatin?
Regions of heterochromatin that mostly contain a high number of tandem repeats (satellite DNA), i.e. centromeres and telomeres are permanently heterochromatic.
67
* What enables X inactivation in mammals?
The self-propagating nature of heterochromatin (can also be problematic when regions of heterochromatin undergo translocations into regions of euchromatin).
In particular, the expression of the Xist gene on both X chromosomes covers regions of DNA, causing it to be silenced.
68
* What is the result of X inactivation?
Regions of the X chromosome are permanently silenced - this is important for gene regulation as if all of the same genes on both of the homologous chromosomes were expressed there would be too many proteins within the cell, so one of the genes must be silenced (different genes between the two chromosomes are silenced, however, not always just one chromosomes becoming completely inactivated).
69
What is a higher order chromatin structure that can affect gene expression?
To silence large stretches of DNA, chromatin can associate/bind to the nuclear lamina through associations with proteins bound to the membrane.
70
How can gene expression be regulated by external messengers?
Hormones and other messengers can either diffuse into the cell (i.e. oestrogen) or bind to receptors on the cell surface, causing the activation of various transcription factors to activate or repress genes.
71
How is RNA polymerase II released from the initial stages of transcription/from the PIC?
This is achieved through the action of a kinase that phosphorylates a characteristic structure at the C-terminal domain, where there are 52 repeats of seven amino acids.
Phosphorylating the serine residues here is vital to end abortive initiation, only then allowing transcription and elongation.
This is achieved by a kinase subunit (CDK7) on the TFIIH transcription factor.
72
How is gene output controlled?
Through the rate of transcription - the higher the rate is, the more protein is produced.
73
Why is such complexity necessary to transcribe eukaryotic genes?
Because a flurry of signals can arrive at the cell at any one time, which require a specific response/the correct cluster of genes to be activated.
Prokaryotic cells are simpler, with less complex responses.
74
What bond is formed between adjacent ribonucleotides by the RNA polymerase enzymes?
Phosphodiester - bases will already be correctly arranged and orientated through complementary interactions with bases on the template strand.
75
What are exons?
Regions of DNA within a gene that code for proteins.
76
What are introns?
Regions of DNA within a gene that do not code for proteins, and need to be removed from pre-mRNA in eukaryotes. >90% of human protein coding genes contain introns.
77
Do prokaryotic genomes contain introns?
No
78
Roughly how much more space in the genome is taken up by introns compared to exons?
10x more space.
79
What is pre-mRNA?
The initial transcript of a gene - contains both introns and exons
80
What is mRNA?
An edited transcript which has had its introns removed through the process of splicing.
In prokaryotes, this is the product of transcription.
Now able to code for a protein.
81
Does splicing occur in prokaryotes?
No, only in eukaryotes
82
How are RNA polymerase II transcripts modified?
Capping of the 5' end
Splicing of introns
Poly(A) tail formed at the 3' end
83
Are all three modifications necessary for mRNA to be able to leave the nucleus?
Yes, or else the transcript would be digested or code incorrectly for a protein.
84
Where do post-transcriptional modifiers associate?
On the heavily phosphorylated C-terminal of the RNA polymerase enzyme.
85
When does pre-mRNA processing occur?
During transcription - it is a 'co-transcriptional' process.
86
What is the first pre-mRNA modifying process to occur?
Capping of the 5' end of the transcript
87
What is 5' capping?
This is where a GMP (guanine monophosphate) molecule is fused to the end of the pre-mRNA transcript in an unusual 5'-5' linkage (reverse orientation) and then methylated.
This is then able to bind to a protein complex called the cap binding complex (CBC), which prevents the action of/untimely degradation by 5' exoribonucleases.
The RNA polymerase enzyme is often stalled until the capping process is complete.
The cap is also involved in the initiation of translation.
88
What are the consequences if 5' capping does not occur?
The transcript will be digested by 5' exoribonucleases and degraded, and even if this does not occur, translation will be unable to be initiated without the presence of this cap.
89
What is the second pre-mRNA modifying process to occur?
Splicing
90
* How were introns and splicing discovered in 1977?
Through studies on adenoviruses, as they discovered that in late stages of infection the mRNA molecules were made up from non-contiguous fragments of the viral genome, indicating that some form of rearrangement had taken place - this solved the difference observed between mRNA molecules in the nuclei of cells and those found in the cytoplasm.
91
What is splicing?
An RNA-mediated trans-esterification reaction resulting in two sequential breakages and re-joining of the sugar-phosphate backbone of the RNA. The result is the excision of introns and fusion of exons.
92
What is a vital feature of ribonucleotides that allows splicing?
The OH group of the 2' carbon (not present on deoxyribose sugars)
93
How many trans-esterification reactions occur during splicing?
Two
94
What is the process of splicing?
First, the 2'OH of a particular adenosine nucleotide which is an essential sequence element in the intron structure directs a nucleophilic attack on the 5'exon/intron border.
Trans-esterification results in a 'lariat intron' structure that is joined with the 3' exon and releases the 5' exon.
In the second stage of splicing, the 3'OH of the 5' exon attacks the intron/3' exon junction. The subsequent trans-esterification fuses the 3' exon and the 5' exon.
95
What is the structure formed out of the intron during splicing called?
An intron lariat (looks like a loop)
96
What is the name of sequences on the pre-mRNA that signal where splicing should occur?
Cis-elements, provides 'intron definition' (the intron/exon boundary).
97
What directs pre-mRNA splicing?
A large complex of proteins and 5 RNA players associate to form a giant complex known as a spliceosome. The RNAs involved are all small nuclear RNAs (snRNAs, U1, U2, U4, U5, U6, the U indicates that the snRNAs are bound to a specific protein, known as SNURPS).
98
How do transesterification reactions occur?
Effectively spontaneously.
99
How precise is a spliceosome?
It has nucleotide precision.
100
What does the spliceosome recognise?
Cis-elements on the pre-mRNA strand
101
What is a continuous sequence of exons called?
An open reading frame (ORF)
102
What is alternative splicing?
This is where exons can be ordered in variable ways, or exons can be skipped during splicing due to variable splice sites. It plays an important role in tissue specific gene expression and brain plasticity.
103
What does alternative splicing allow?
The expansion of the proteome, allowing several proteins to be formed from just one gene.
104
How does alternate splicing occur?
Proteins that associate with certain sequences along the pre-mRNA molecule can cause the strengthening or weakening of various splice sites, therefore regulating the exclusion and/or inclusion of exons.
105
What effect do enhancers have on splicing?
They make it more likely that the spliceosome/splicing factors will recognise the 3' and 5' sites that flank that exon, preventing 'skipping' and excision of the exon.
106
What effect do negative factors have on splicing?
These can be tissue specific but prevent or lessen the chance of splicing factors/spliceosomes recognising the 3' and 5' sites that flank the exon, making it more likely to be skipped and excised. Results in potential exclusion.
107
* What is an example of the power of alternative splicing?
In drosophila, alternate splicing can result in more than 38000 DSCAM isoforms - this diversity is likely to contribute to the specificity of neuronal connectivity.
108
What is the relationship between complexity of the organism and number of genes and/or introns?
As complexity increases, so does the number of genes and introns.
109
What is the final process of pre-mRNA processing?
3' end processing - the addition of a poly(A) tail.
110
What directs cleavage and polyadenylation?
Bipartite (two separate) cis-regulatory elements.
111
What is the first signal for cleavage and polyadenylation?
A hexamer (AATAAA / AAUAAA) located about 30b upstream of the actual cleavage and polyadenylation site.
112
What is the second signal for polyadenylation?
A region rich with GT / GU bases (mostly uracil) known as the downstream sequence element (DSE), that lies after the cleavage site, and is still transcribed by the polymerase enzyme until a stop codon is reached.
113
What are the two steps of polyadenylation?
1. Cleavage
| 2. Subsequent polyadenylation of the product
114
Where does cleavage and polyadenylation machinery associate?
At the AAUAAA and at the DSE on the pre-mRNA, aka 'Poly(A) sites' present at the 3' ends of the transcript.
115
What occurs after cleavage?
The poly(A) site on the 3' end of the pre-mRNA has a poly(A) tail added on to the end by a poly(A) polymerase enzyme (PAP) in a reaction that doesn't require a template. This tail is then covered by poly(A) binding proteins (PABP)
116
What does polyadenylation create?
A uniform 3' end that is found on all protein-coding mRNAs.
117
Why is polyadenylation necessary?
It is critical for nuclear cytoplasmic export, stability and translatability of the mRNA. Other proteins are able to recognise the poly(A) binding proteins (PABP).
118
What exists either side of the open reading frame (ORF) on mature mRNA?
3' and 5' untranslated regions, which contain potentially thousands of nucleotides that don't code for anything.
119
How are mature mRNA molecules protected from the premature degradation by mRNA hydrolases/exonucleases?
By the 5' cap and the 3' poly(A) tail.
120
Diseases that affect pre-mRNA processing most commonly effect:
Splicing patterns or splicing efficiency of a particular exon (i.e. mutations in dystrophin gene cause Duchenne's muscular dystrophy due to faulty protein formation as splice sites are mutated/changed).
121
What can mutations at splice sites cause?
Reduced or abolished splicing of particular introns, faulty pieces of mRNA that contain intronic sequences and therefore produce faulty proteins, changing of protein message or creation of a premature stop codon.
If the protein output is changed, this can result in disease.
Mutations affecting splice factors can have a similarly wide range of effects.
122
What can mutations affecting poly(A) signals cause?
This can reduce or increase cleavage and polyadenylation, affecting the total output of mRNA.
123
* How is cardiac hypertrophy related to splicing?
Mutations that occur at the 3' splice site can result in exon skipping - i.e. a 5 base pair deletion in the intron 3 of the TNNT2 gene forces exon 4 to be skipped, as a splice site is now unrecognisable. This causes cardiac cells to become hypertrophic (increased growth and therefore size).
124
What happens if there is a point mutation within the hexamer signal prior to the cleavage site?
The signal cannot be recognised by poly(A) machinery, causing there to be reduced efficiency and a reduction in cleaved mRNA, resulting in a drop in output.
* This is seen is thalassemia, when this mutation occurs in the alpha-globin gene, causing a reduction in the beta globin protein. This means that there is an imbalance/excess of alpha chains, resulting in an aggregation of these alpha chains in the red blood cells, affecting their function and eventually resulting in anaemia.
125
What is alternative polyadenylation (APA)?
This is where RNA molecules with different poly(A) tails arise from the same gene. The production of distinct poly(A) sites can be done through alternative splicing mechanisms (different regions left on the 3' end of the mRNA molecule), regulation through the action of RNA processing proteins and also through transcriptional dynamics.
126
What breaks down the introns after splicing?
Degrading enzymes such as exonucleases.
127
What is a unit of nucleic acid?
A trinucleotide codon
128
What is a unit of a protein?
An amino acid
129
What is the ratio between codons and amino acids?
1:1
130
How are instructions in RNA decoded?
Through tRNA molecules - they are bonded to amino acids (1 per tRNA molecule) and have trinucleotide sequences known as anticodons that are complementary to codons on the mRNA strand.
131
What is the meaning of degenerate code?
This means that multiple codes can code for the same thing - i.e. multiple codons code for the same amino acid.
132
What is always the first amino acid and the sequence of the start codon?
Methionine, and it is only coded for by a specific start codon, AUG. Translation always starts from this codon.
133
What are 'stop' codons?
These are regions not recognised by tRNA molecules an do not code for proteins. They are UAA, UAG and UGA. These are instead recognised by proteins, which force the termination of translation.
134
How many reading frames does each nucleic acid have?
Each has 3 possible configurations that can be assigned, that can result in three drastically different protein configurations - the bad effects of these can be seen in frame shift mutations.
135
How are triplets/codons read?
In a non-overlapping fashion from a start point - the start codon AUG.
136
By which process is the correct start point picked out?
Translation initiation.
137
What is the open reading frame (ORF)?
A continued region after the start codon that contains exclusively triplets that encode for amino acids.
138
What happens if a point mutation causes the formation of a stop codon?
Translation is prematurely halted, causing a shortened protein with a faulty or aberrant function to be formed. This is a nonsense mutation.
139
What is a missense mutation?
This is where a single point mutation occurs/a single base is changed. Due to degenerate code, this can result in the association of the same amino acid in a silent mutation, but can cause the association of a different amino acid which may affect protein function in a positive or a negative manner.
140
What is required for the translation of mRNAs?
tRNAs (transfer RNAs) act as a physical representation of the 1:1 relationship between amino acids and codons by transferring the amino acids to the ribosome-mRNA complex
Ribosomes orientate amino acids and catalyse the formation of a polypeptide chain
Translation factors (proteins) are necessary for initiation, elongation and termination, phosphorylation of these factors can be vital for their function and gene expression.
A lot of energy is released from GTP (guanine triphosphate) as the formation of peptide bonds requires a lot of energy to occur.
141
What shape does a tRNA molecule resemble?
A clover leaf
142
At which end is the amino acid attached to a tRNA molecule?
The 3' end via an ester/amino-acyl bond
143
What are the names of the three loops on a tRNA molecule, from left to right/5' to 3'?
The D loop, the anticodon loop (contains trinucleotide complementary to codon on mRNA) and the T loop.
144
How specific are tRNA synthetases?
Highly specific - they will only associate with their target amino acid and tRNA molecule with the correct anticodon (although this may be multiple anticodons due to degenerate code). The process is also 'proof reading' to ensure the accuracy of the process.
145
What is the structure of a ribosome?
They consist of both protein and rRNA components - the RNA is the catalytic component, forming the ribozyme (an RNA molecule able to act as an enzyme).
146
How many subunits make up both prokaryotic and eukaryotic ribosomes?
2 (50s and 30s in prokaryotic, 60s and 40s in eukaryotic)
147
What is the classification of prokaryotic ribosomes?
70s, made up of a 50s and 30s subunit.
148
What is the classification of eukaryotic ribosomes?
80s, made up of a 60s and a 40s subunit.
149
Where are eukaryotic ribosomes processed?
The ribosomal subunits are assembled in the nucleolus and exported.
150
How does 'de-coding' work?
The tRNA molecule recognises a codon on the mRNA molecule that corresponds to its anticodon via base pairing. The base pairing occurs between the nucleotides in the anticodon codon respectively.
151
What is the role of the third base in the anticodon?
As the number of potential codons formed by AUGC is 4^3 (64), 61 tRNA molecules would be necessary (as there are 3 stop codons). However, only 45 are seen in most organisms, meaning that some must be able to recognise multiple anticodons - this is achieved through differences in the third base. Base pairings at this third base, or the 'wobble position' are less strict, and a base can pair with more than one complementary base.
This 'wobble hypothesis' was proposed by Francis Crick.
For example, a wobbling third base allows the four codons that correspond to an alanine amino acid to be recognised by only two tRNAs, G forms base pairs with both C and U in this situation (only one hydrogen bond formed).
152
What bond is formed between amino acids?
Peptide bond
153
Can tRNA molecules be reused?
Yes - once the peptide bond has been formed between two amino acids, the tRNA molecule is released from its peptidyl linkage and diffuses away.
154
What are the three sites on a 60s ribosomal subunit to accommodate tRNA molecules?
E site - exit site
P site - peptidyl site
A site - aminoacyl site
155
How does translation initiate and elongate?
The P site is occupied by the methionine initiator tRNA. The A site is lined up with the next codon and is ready to accommodate the corresponding tRNA molecule.
Once the correct tRNA molecule has entered this site, the peptidyl transferase activity catalyses peptide bond formation.
The ribosome shifts one codon along, releasing the initiator tRNA through the E site, the tRNA with the growing peptide is positioned at the P site and then the A site is open for the next complementary tRNA molecule to occupy it.
This process repeats until a stop codon is reached.
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What enzyme catalyses the synthesis of peptide bonds between adjacent amino acids?
Peptidyl transferase
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How many tRNA molecules can the ribosome deal with at one time?
3, if you include the one being removed at the E site - the ribosome can only really hold two in position within the complex.
158
What happens at the stop codon?
There is no complementary tRNA molecule but rather proteins associate here that trigger the splitting of the two ribosomal subunits from each other and their dissociation from the mRNA molecule. The peptide is also released, now fully formed.
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Can multiple ribosomal complexes act on one mRNA transcript at once?
Yes, each coding their own protein. They will be moving in the same direction but will have started at different times.
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What is the role of the cap and the poly(A) tail in translation?
Cap binding proteins and proteins that bind to the poly(A) tail (PABP) interact with each other to form a circular mRNA molecule. The cap and the poly(A) structure recruit the ribosome to the mRNA, acting as translation initiation factors.
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How does the 5' cap play a key role in translation initiation for eukaryotes?
The cap protein allows for the small ribosomal subunit to be recruited to the mRNA strand at the cap where it can then 'scan' the mRNA for a start (AUG) codon. Once the start codon has been identified, the large ribosomal subunit can associate with the smaller an translation can commence. The cap protein acts as an anchor for this smaller subunit, allowing function.
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How can translation be regulated?
Phosphorylation of transcription initiation factors
| Through sequences that are present on the mRNA template
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Why is it important to regulate translation?
Because the cell invests a lot of energy into the translational machinery and translating all the necessary proteins, so energy must be conserved where possible.
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* What is an example of translational regulation via phosphorylation of translation initiation factors?
4E binding proteins (4E-BP) are able to bind to a specific cap protein (eIF4E) and block interactions between this and other cap binding proteins (such as eIF4G). This prevents the formation of a functional cap and therefore also the association of the small ribosomal subunit to the mRNA.
Phosphorylation of 4E-BP by MAP kinases in response to insulin (ras/raf pathway) causes the protein to become inactive, allowing the functional cap to be formed and therefore the recruitment of a ribosome and translation.
165
What are UTRs?
Untranslated regions - sequences on mRNA molecules that are not translated into proteins. Present at the 3' and 5' ends.
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How can untranslated regions/UTRs act as regulatory targets?
The UTRs can contain important regulatory sequences that affect translation and influence the fate of mRNA within the cell.
For example, regulation of intracellular iron:
If high concentrations of Fe are present, they bind to iron responsive proteins (IRPs) and cause a conformational change that prevents association with mRNA molecules/sequences in the 5' UTR of a gene for a protein that regulates iron storage. Due to the lack of binding, translation occurs for the mRNA, increasing ferritin storage protein (FER) levels that will result in iron storage (to avoid toxic levels being reached) and transport out of the cell.
In low concentrations of iron or moments of oxidative stress, no Fe atoms bind to the regulative protein so these are able to bind to the mRNA transcript and inhibit the initiation of translation and therefore production of proteins that would carry out iron storage, allowing more to be available to the cell.
This process causes fast responses at a transcriptional level and is highly regulated as deficiencies could result in anaemia.
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How can translation recognise faulty mRNA strands?
Once splicing is complete, exon-exon junctions are marked by the deposition of exon junction proteins/complexes (EJC) that can serve as markers to identify faulty mRNAs that contain premature stop codons. The presence of an EJC downstream of a stop codon indicates a faulty mRNA and induces its degradation. Efficient translation is therefore prevented.
168
Why is it difficult to find mutations from studying mRNAs?
Because faulty mRNAs are likely to be degraded prior to detection.
169
How can the half life of mRNA effect gene expression?
The half life dictates for how long proteins are able to be translated from the molecule, so shorter half lives result in smaller changes to protein concentration as exposure time is decreased. The comparatively short half lives of mRNA molecules prevent responses from stimuli for occurring for longer than necessary, increasing ability and speed of response.
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At what points can gene expression be regulated?
Transcriptional control: chromatin structure, transcription factors (activators/repressors), initiation of transcription
Pre-mRNA processing: capping of 5' end, poly(A) tail on 3' end, splicing, alternate splice sites, alternate poly(A) sites - mostly co- but some post-transcriptional
mRNA export and localisation: time taken to export from the nucleus can be a method of regulation, mRNA can be localised with the help of other small, non-coding RNA molecules (which can then bring them into closer proximity with RNAi molecules)
Translation regulations: translation initiation factors and phosphorylation of
Degradation of mRNAs and mRNA halflife time
171
* Which cellular machineries can antibiotics target in prokaryotes?
Inhibition of DNA replication (quinolones)
Inhibition of transcription (rifamycin)
Inhibition of translation (tetracycline, erythromycin, streptomycin, chloramphenicol)
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* How do aminoglycosides inhibit translation in prokaryotes?
Through changing the shape of the 30s ribosomal subunit, causing mRNA to be misread.
173
* How does tetracycline inhibit translation in prokaryotes?
This drug blocks ribosome docking site of tRNA (A site).
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* How does chloramphenicol inhibit translation in prokaryotes?
This drug inhibits peptide bond formation.
175
* How does macrolide inhibit translation in prokaryotes?
This drug binds to the 50s subunit and prevents mRNA from moving through the ribosome.
