From DNA to Protein in 2 Hours Flashcards
Revision (70 cards)
1
Q
What is the genome?
A
The total DNA in each cell constitutes the genome.
2
Q
What is the role of the genome?
A
The genome carries the genetic information required for making the whole organism.
This information is stored in the nucleotide sequence of the genome.
3
Q
What determines the amino acid sequence of polypeptide chains?
A
The DNA nucleotide sequence.
4
Q
How is information transmitted?
A
Via the intermediate RNA
5
Q
What Is the process from DNA to protein?
A
DNA is transcribed (the change of one nucleotide to another nucleotide) to form RNA.
The RNA is then translated (Nucleotide to amino acid) to form protein.
6
Q
What is the central dogma?
A
The flow of the process from DNA to protein.
7
Q
Eukaryotic means what?
A
That the cell has a nucleus.
8
Q
Nomenclature
A
Base Nucleoside Adenine Adenosine Cytosine Cytidine Guanine Guanosine Thymine Thymidine Uracil Uridine
9
Q
What is Polymerisation?
A
You can only extend an existing nucleotide chain
by adding a phosphate to a free 3” phosphate group.
A phosphodiester bond is formed between a free 3’ OH group and a 5’ triphosphate. This consumes 2 high-energy bonds.
The energy in ATP is in the bonds between the negatively charged phosphate groups. When the bonds break, the energy is released.
10
Q
What is the name of the sugar in RNA and what is the name of the sugar in DNA?
A
Ribose in RNA and Deoxyribose in DNA.
11
Q
What is attached to carbon 1 and what is attached to carbon 5?
A
The base is attached to carbon 1 and phosphate is attached to carbon 5.
12
Q
What is nucleoside composed of?
A
base + sugar
13
Q
What is a nucleotide composed of?
A
nucleoside + phosphate group(s)
14
Q
What are the bases in RNA and DNA?
A
The bases in DNA are:
A,C,G,T
The bases is RNA are:
A,C,G,U
15
Q
What bases are classed as purines?
A
Adenine (A)
Guanine (G)
16
Q
What bases are classed as pyrimidines?
A
Uracil (U)
Thymine (T)
Cytosine (C)
17
Q
What are the DNA building blocks?
A
dATP dCTP dGTP dTTP
deoxy-adenosine-triphosphate etc.
18
Q
What are the RNA building blocks?
A
ATP CTP GTP UTP
-adenosine-triphosphate etc.
19
Q
How do you know that nucleic acids have a direction?
A
New nucleotides are only added to a free 3’ end.
DNA and RNA can only be extended in a 3’ to 5’ direction. You can read them in a 5’ to 3’ direction. You read the base e.g. C, A, G.
20
Q
How do nucleotide analogues act as drugs?
A
ZDV (zidovudine) = AZT (azidothymidine) = Retrovir
Analogue of thymidine
This is incorporated into the growing viral DNA.
It lacks the 3’ OH group, therefore chain elongation is terminated.
It only works because viral revers transcriptase has higher affinity for ZDV than human DNA polymerase.
(It looks like thymidine but doesn’t have a free 3’ group).
21
Q
What is the structure of the double helix and the base pairs?
A
DNA consists of 2 nucleotide strands
- one 5’ to 3’
- one 3’ to 5’
They are antiparallel and complimentary.
Each nucleotide strand has a sugar-phosphate backbone on the outside.
Base pairs are located on the inside of the strand and the 2 base pairs are hydrogen bonded together.
22
Q
When is DNA replicated and why?
A
DNA has to be replicated (duplicated) before cell division so that the daughter cells have a complete complement of the genome.
23
Q
How is the replication of DNA described and what is it catalysed by?
A
It is semi-conservative and catalysed by DNA polymerase.
24
Q
What are the restrictions of DNA polymerase?
A
It can only add to existing nucleic acids.
It cannot start DNA synthesis on their own.
It requires an RNA primer to start replication.
25
How many origins of replication do eukaryotic cells have and what is the function of them?
Eukaryotic genomes have many origins of replication.
This allows replication to start simultaneously at several points in the genome.
It is also bidirectional and ensures that replication can be finished in a reasonable time.
26
Why is replication continuous on one strand and discontinuous on the other?
Nucleotides cn only be added to free 3' ends.
The leading strand always has a free 3' end. On the leading strand DNA polymerase elongates both strands. Single-strand binding proteins keep the template strands separated.
The other strand has to be replicated in short segments. This is called the lagging strand and the short segments are called okazaki fragments.
Primase synthesises a primer and DNA helicase unwinds the double helix.
27
What are the major points of DNA replication?
dATP, dTTP, dCTP as building blocks
- one phosphate group forms phosphodiester bond.
- two leave as PPi (pyrophosphate) - energy supply
Helicase needed to unwind helix (and stop it rewinding)
Replication fork with leading (3' to 5') and lagging (5' to 3') template strands forms.
Copying leading strand template in 5' to 3' direction leaves free 3' end for next nucleotide.
Copying lagging strand template is more complex - okazaki fragments.
DNA synthesis needs an RNA primer - synthesized by primase.
28
How does the body proofread and repair mistakes?
Incorporating the wrong nucleotide can create mutations
- can be deleterious
- errors occur once every 10to the power of 4 to 10 to the power of 5 base pairs.
DNA polymerase has 3' to 5' exonuclease activity
- removes incorrect nucleotide
- improves error rate to one in 10 to the power of 9 to 10 to the power of 10 base pairs.
Further repair systems exist.
29
What are features of RNA?
It is usually single stranded.
Can contain local stretches of intramolecular base-pairing. In regions where the bases are complementary, RNA creates stems. Where the base pairs are not complementary, loops are created.
RNA contains U (Uracil) instead of T (Thymine).
30
What are the 3 main classes of RNA?
```
Ribosomal RNA (rRNA) combines with proteins to form ribosomes where protein synthesis takes place.
Transfer RNA (tRNA) carries the amino acids to be incorporated into the protein.
Messenger RNA (mRNA) carries the genetic information for protein synthesis.
Ribosomal and transfer RNA are stable. Messenger RNA is unstable.
```
31
What is the structure and role of tRNA?
It acts as adapters between nucleic acid code and amino acid code.
Anticodon consists of 3 nucleotides.
Specific amino acids are attached to the 3' end
- Depends on anticodon sequence.
All tRNA molecules have a distinct 3D structure.
When flattened into a 2D structure, it appears as a cloverleaf structure.
32
What is the role of RNA polymerase?
RNA polymerase is a multi-subunit complex.
| RNA polymerase uses one DNA strand as a template to copy the nucleotide sequence into RNA.
33
In what direction do RNA and DNA strands run?
RNA will also run antiparallel to the DNA. As it is made, its synthesized in this direction.
This strand is called a template strand.
You may also have a coding strand which is an almost identical sequence as the RNA.
34
What are the steps of transcription?
RNA polymerase binds
- it detects initiation sites by promotors on DNA
- It requires transcription factors to bind
The DNA chain then separates
- local unwinding of DNA
- to gain access to the nucleotide sequence
Transcription Initiation
- Selection of the first nucleotide of the growing RNA
Elongation
- addition of further nucleotides to the RNA chain
Termination
- release of finished RNA.
(The RNA polymerase moves in a 3' to 5' direction along the template DNA strand and creates a strand of RNA complementary to the template strand and almost identical to the coding DNA strand other than that T is substituted with U and ribose is used instead of deoxyribose).
35
what is the role of TBP (TATA box binding protein) and the transcription factor that it is a part of?
TATA box binding protein
TBP recognises TATA box.
All polymerase molecules require the binding of TFIID (Transcription Factor IID)
- TFIID is a general transcription factor
- Required for all Pol II transcribed genes.
TFIID introduces a kink into DNA
- determines transcriptional start and direction.
This provides a landing platform for further transcription factors and for RNA polymerase.
36
How is transcription initiated?
The initiation of transcription requires additional general transcription factors.
There is a precise order of assembly.
Pol II and TFIIF extend the transcript on their own.
TFIID remains at promotor, so that a new initiation complex can assemble. This allows transcription at low, basal rates.
(Where TATA boxes are available there will be a low rate of transcription).
1. The first transcription factor, TFIID, binds to the promotor a the TATA box.
2. and another transcription factor joins it.
3. RNA polymerase II binds only after several transcription factors are already bound to the DNA.
4. More transcription factors are added.
5. and the RNA polymerase is ready to transcribe RNA.
37
How is transcription elongated?
A transcription "bubble" moves in one direction along the DNA.
DNA is unwound in front of the polymerase, and rewound behind it.
RNA chain is synthesised in a 5' to 3' direction.
New RNA sequence is complementary to the template strand.
It's identical to the coding strand
- but U instead of T
38
How is transcription terminated?
Newly synthesised RNA makes a stem-loop structure
- followed by a stretch of Us
A specific enzyme cleaves the (now finished ) RNA
- RNA is released
- Polymerase dissociates
39
How is transcription regulated?
Transcription requires "specific" transcription factors
- DNA-binding proteins
- Contain 2 functional domains which can be physically separated. These are the DNA-binding domain and the Transcriptional activation domain.
Enhancers are proteins that bind to specific DNA sequences in the vicinity of a promotor.
Regulate transcription positively or negatively.
A long stretch of DNA lies between the activator binding site and the transcription complex.
DNA bending can bring an activator protein, bound to an enhancer element far from the promotor, into contact with the transcription complex.
40
How is coordinated gene expression carried out?
A stressor (e.g. drought) activates transcription of a regulatory protein through a drought-sensitive transcription factor.
Binding or the regulatory protein to the stress response element (SRE) stimulates transcription of genes A,B and C which produce different proteins participating in the stress response.
This occurs in response to a specific stimuli e.g. hormones, cellular stress etc.
41
What are steroid receptors and how do they carry out their role?
Steroid receptors are part of a family of transcription factors, a subset of a much larger family of nuclear hormone receptors.
They are structurally similar, as the DNA-binding and ligand-binding domains are highly conserved (appear in almost everything).
It's located in the cell cytoplasm (inactive).
On binding to a ligand (steroid) move to nucleus and bind to DNA at steroid-response elements (SREs).
42
How is gene regulation carried out in the Glucocorticoid receptor?
Steroids are transported in blood
- bound to albumin or specific transport proteins.
Free steroids enter target cells by diffusion.
Bind to inactive steroid receptors in cytoplasm.
This activates the receptor.
Which translocates to the nucleus.
Where it binds to response elements
- usually as a homodimer (a protein composed of two polypeptide chains that are identical in the order, number, and kind of their amino acid residues).
Coordinated regulation of a set of genes.
43
Eukaryotic genes have noncoding sequences.
| What are these regions called, what is the range in the number that can appear on a gene and how are they removed?
```
Coding regions (exons) are interrupted by non-coding regions (introns)
-There are between 1 and more than 50 introns per gene.
Exons and introns are transcribed together.
Introns have to be removed before translation into protein.
- through splicing.
```
44
How are the ends of mRNA processed?
A "cap" of modified GTP is added at the 5' end of pre-mRNA. The coding sequence of the pre-mRNA is recognized and cut by an enzyme.
In mature mRNA a poly A "tail" is added at the 3' end.
(Addition of poly(A) tail. Addition of 5' cap).
45
How are transcription and translation are compartmentalised in eukaryotic cells?
The DNA never comes out of the nucleus to be transcribed. But is translated in the cytoplasm by ribosomes.
46
What are codons and anti-codons composed of and how do they relate to each other?
During translation, anticodons of tRNA molecules form base pairs with codons on mRNA.
Codons and anticodons each consist of 3 nucleotides.
Therefore, the genetic code is based on triplets.
4 different nucleotides available
- 64 possible combinations
code for 20 amino acids
47
Why can the genetic code be described as both degenerate and unambiguous?
You have start and stop codons in your DNA.
Degenerate (A code in which several code words have the same meaning. The genetic code is degenerate because there are many instances in which different codons specify the same amino acid)
- many amino acids have more than one codon.
Unambiguous
- each codon codes for only one amino acid (or a stop).
Nearly universal.
48
How can Each RNA molecule be translated through reading frames?
Each RNA molecule can be translated in 3 different reading frames
- depends on where translation starts.
Each RNA molecule can be translated using 3 different frames.
49
What are the components of translation?
Translation requires:
- Amino acids
- tRNAs
- Aminoacyl-tRNA synthetase (also called tRNA-ligase, is an enzyme that attaches the appropriate amino acid onto its tRNA)
- A specific set of protein factors for the initiation of protein synthesis, elongation of polypeptide chain and translocation and finally termination.
- ATP and GTP as sources of energy
- Ribosomes
- mRNA
50
What is the role of Aminoacyl-tRNA synthetase?
Aminoacyl-tRNA synthesase bind amino acids to their corresponding tRNA molecule(s)
- highly specific.
At least one for each amino acid.
ATP provides energy for formation of covalent bond.
51
Ribosomes have 3 tRNA binding sites what are their names and in what direction does the tRNA molecule move from one site to another?
E-Exit
P-Peptidyl
A-Aminoacyl
THe tRNA moves from the A site to the P site to the E site
52
What is the process of initiation?
Requires initiation factors (IFs).
GTP is hydrolysed to provide energy for initiation.
A small ribosomal subunit binds to 5' end of mRNA.
Moves along the mRNA until AUG (start codon) is found (ATP-dependent).
Special "initiator" tRNA with an UAC anticodon base, pairs with the start codon
- carries methionine.
Large subunit joins assembly and initiator tRNA is located in P site.
53
What is the process of Elongation?
AN elongation factor (EF-1alpha), brings the next aminoacyl-tRNA to the A site.
- anticodon (here: CGU) base-pairs with codon (here: GCA).
GTP is hydrolysed, EF is released from tRNA.
A second elongation factor (EFbetay) regenerates EF1alpha to pick up the next aminoacyl-tRNA.
54
How is peptide bond formation and translocation carried out?
Peptidyl transferase catalyses peptide bond formation between amino acids in the P and A sites
- peptide now located in A site.
Elongation factor EF-2 moves ribosome along the mRNA
- by one triplet.
"Empty" tRNA moves to E site
- can exit and become reloaded with an amino acid.
tRNA with the growing peptide moves from the A to the P site.
A site is free for the next aminoacyl-tRNA.
55
How is termination carried out?
Occurs when the A site of the ribosomes encounters a stop codon
-UAA, UAG or UGA.
No aminacyl-tRNA base-pairs with stop codons.
Release factor RF binds to the stop codon
-GTP hydrolysis.
Finished protein is cleaved off tRNA.
The components -rRNA, mRNA and tRNA - dissociate from one another.
Whole process starts all over again with small subunits being bound by IF ready for translation of a new protein.
56
What different types of base mutations can occur?
Point numation
- change is a single base in DNA
MIssense mutation
- results in a change of amino acid sequence
- can change protein function e.g. altered haemoglobin in sickle cell anemia
Nonsense mutation
- creates a new termination codon
- changes length of protein due to premature stop of translation
Silent Mutation
- no change of amino acid sequence
- due to degeneracy of the genetic code
- no effect on protein function
Framshift Mutation
- addition or deletion of a single base (or 2)
- Changes reading frame of translation into protein.
57
What are the different types of chromosomal mutations?
```
Chromosomal mutations
- affect larger portions of the genome
Deletions
Duplicatios
Inversions
Translocations
```
58
What happens with the finished protein?
Targeting
- moving a protein to it's final cellular destination
- many possible locations within a cell
- depends on the presence of specific amino acid sequences within the translated protein
Modification
- addition of further functional chemical groups
Degradation
- unwanted or damaged proteins have to be removed
59
Where are the proteins made by "Free" and "Bound" ribosomes destined for?
```
Free ribosomes in the cytosol make proteins destined for
- cytosol
- nucleus
- mitochondria
- translocated post-translationally
Bound ribosomes on the rough ER make proteins destined for
- plasma membrane
- ER
- Golgi apparatus
- Secretion
- translocated co-translationally
```
60
What Post-translational modifications are possible?
Glycosylation - addition and processing of carbohydrates in the ER and the Golgi (adding sugars is important for targeting and recognition).
Formation of disulphide bonds in the ER.
Folding and assembly of multisubunit proteins in the ER.
Specific proteolytic cleavage in the ER, Golgi and secretoyry vesicles.
Hereditary form of emphysema results from misfolding f the protein alpha1-antitrypsin in the ER.
(Proteolysis - Cleaving the polypeptide allows the fragments to fold into different shapes).
(Phosphorylation - Added phosphate groups alter the shape of the protein).
61
What is an example that shows the clinical relevance of protein targeting?
I-cell disease (mucolipidoses II)
- inherited recessive disorder of protein targeting
- proteins normally destined for lysosomes are not properly sorted in the Golgi
- end up secreted from the cell
- lysosomes can not properly digest material, become clogged.
- death before age 8
62
How many different types of RNA polymerase do prokaryote and Eukaryote cells contain and how can they be differentiated?
Prokaryotic cells have one type of RNA polymerase.
Eukaryotic cells have 3 types of RNA polymerase
- Pol I, Pol II, Pol III
- Can be distinguished by their sensitivity to toxins like alpha-amanitin (which is derived from a fungus)
- Pol II synthesises all mRNA.
63
How does RNA polymerase bind to promoters?
RNA Pol II has a specific promotor.
Transcription starts at nucleotide +1.
TATA box is present about 25 nucleotides before the transcriptional start (-25)
64
What is a homodimer?
Homodimer: a protein composed of two polypeptide chains that are identical in the order, number, and kind of their amino acid residues
65
How many rRNA molecules do ribosomes contain?
Ribosome contain 4 rRNA molecules
| - 3 in bacteria
66
How many protein components do ribosomes contain specifically in prokaryotic cells?
55 polypeptides in prokaryotic cells.
67
What is the polysome?
a complex of ribosomes strung along a single strand of messenger RNA that translates the genetic information coded in the messenger RNA during protein synthesis
68
What is the TATA box?
The TATA box is a sequence of DNA found in the core promoter region of genes.
69
What is the role of a transcription factor?
In molecular biology, a transcription factor (TF) is a protein that controls the rate of transcription of genetic information from DNA to mRNA, by binding to a specific DNA sequence. The function of TFs is to regulate—turn on and off—genes in order to make sure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism.
70
What is the role of TFIID (Transcription Factor II D)?
(TFIID) is one of several general transcription factors that make up RNA polymerase II. Before the start of transcription, TBP (TATA box binding protein) a part of the (TFIID) complex binds to the TATA box in the core promoter of the gene.
