Genes & Replication Flashcards
What is the structure of an amino acid?
Define Chirality
Chirality means “handed”
The central carbon of an amino acid is the chiral centre, it has four substitutents bound to it giving rise to different isomers.
Describe the key features of this peptide (amino terminus, carboxyl terminus, peptide bond & side chain)
List the different bonds that hold protein together in their conformations
- Covalent Bonds
- Hydrgen Bonds
- Van der Waals Forces
- Ionic Interactions
- Hydrophobic Interactions
What is a covalent bond?
Bond where two atoms share electrons - it is the strongest type of bond
What is a hydrogen bond?
When two atoms bearing partial negative charges share a partially positively charged hydrogen, the atoms are engaged in a hydrogen bond
What is an ionic interaction?
These arise from the electrostatic attraction between charged side chains
What are Van der Waals forces?
These are transient, weak electrostatic attractions between two atoms, due to the fluctuating electron cloud surrounding each atom which has a temporary electric dipole
What are hydrophoic interactions
The major force driving the folding of proteins into their correct conformation.
They create a hydrophobic core and a hydrophilic surface to the majority of proteins
Describe the four folding structures of a protein
- Primary - linear sequence of amino acids
- Secondary - local structural motifs
- Tertiary - secondary structural motifs - protein folds into compact domains
- Quaternary - 3D shape of a multimeric protein
What are the two major secondary structures of a protein?
- Alpha helices
- Beta-pleated sheets
Define a nucleic acid
DNA and RNA are examples of nucleic acids
Nucleic acids are macromolecules made up of nucleotides
Define a nucleotide
A nucleotide is composed of a base, a sugar, and a phosphate group.
Give some examples of a nucleotide
- Deoxyadenosine 5’-triphosphate (dATP)
- Adenosine monophosphate (AMP)
Define a nucleoside
Composed of a base + sugar
(i.e. no phosphates)
Give some examples of a nucleoside
- (deoxy)cytidine
- (deoxy)thymidine
- (deoxy)uridine
- (deoxy)adenosine
- (deoxy)guanosine
What are the Bases? (inc. DNA and RNA)
- Cytosine (C)
- Thymine (T) (DNA only)
- Uracil (U) (RNA only)
- Adenine (A)
- Guanine (G)
What is the difference between a purine and a pyrimidine base?
Purines = one carbon ring nitrogen base
Pyrimidines = two carbon ring nitrogen base
Which bases are purines?
Which bases are pyrimidines?
- C - pyrimidine
- T - pyrimidine
- U - pyrimidine
- A - purine
- G - purine
Describe the primary structure of DNA
- linear sequence of bases
- long chain of deoxyribose units linked by phosphodiester links
- Phosphate groups are attached to the 5’- and 3’-carbon atoms of each sugar to form the backbone chain of DNA.
- 5’ and 3’ ends (which face opposing directions)
- 5’ end carries a free phosphate
- 3’ end carries a free hydroxyl (-OH) group a
Describe the secondary structure of DNA
- Right-sided double helix
- Two chains in the helix run in opposite directions
- The two chains are held together by hydrogen bonds between the bases (this creates a major and minor groove)
- The two strands are complementary in their sequence due to the specificity of base pairing
Explain Watson-Crick base pairing
The bases of DNA always pair specifically -
A – T
C – G
How main hydrogen bonds are in each base pair (A/T and C/G)
- A/T = 2 therefore it is less stable
- C/G = 3 therefore it is more stable
What are the key differences between the E. coli (bacterial) and human (eukaryotic) genome?
- Size - human genome is larger
- Human genome divided into chromosmes
- E coli genome single circular DNA molecule
What is the purpose of the nucleosome?
Describe its structure.
The nucleosome enables the packaging of DNA into smaller molecules.
Structure:
- 8 histones
- DNA wrapped around histones
Describe the human karyotype
The human karyotype:
- 22 pairs of autosomal chromosomes and one pair of sex chromosomes
- Karyotype - the number and visual appearance of the chromosomes in the cell nuclei of an organism or species*
What are the typical START and STOP codons?
Start = methionine (AUG)
Stop = cysteine (TGA)
Define semi-conservative replication
DNA replication is semi-conservative
- Each strand forms the template for a new strand of DNA (i.e. one strand inherited from parental DNA, the other is newly synthesised)
- The two strands are complementary to each other so each strand serves as a template for the synthesis of the other strand.
- Replication generates two identical copies
What is the role of DNA helicase?
Unwinds DNA
The DNA double helix is very strong/stable but it must be broken for DNA to replicate - DNA helicase is the enzyme that breaks the hydrogen bonds between the base pairs enabling access for the transcription machinery.
NB: this requires ATP.
What is the role of DNA polymerase?
What is required for DNA polyermase to perform its function?
DNA polymerase synthesise new DNA
This requires primers for the polymerase to extend from - the initial segments are synthesised by DNA primase and elongated by DNA polymerase
Describe the reaction catalysed by DNA polymerase
- DNA polymerases add nucleotides to the 3’ end of a growing chain.
- DNA polymerases require a template strand, an olinucleotide primer, and a supply of of deoxynucleotide triphosphates (dNTPs)
- DNA (and RNA) synthesis occurs in 5’ to 3’ direction.
- Energy drives the reaction
- A free 3’ hydroxyl group is required.
What is a nucleoside analgoue and how can it be used therapeutically?
Nucleoside analogues are pharmacological compounds that mimick nucleosides.
They can be used pharmacologically (eg: HIV treatments) as chain terminators
What is the replication fork?
**Replication fork = site of DNA synthesis **
It is an asymmetric molecule because DNA synthesis is occuring in opposing directions (the templates for the two new daughter strands have opposite orientations: 3’ to 5’ and 5’ to 3’)
*Remember - DNA synthesis can only occur at discrete points on the DNA molecule *
What do the terms leading strand and lagging strand mean?
Remember - replication occurs within the Replication Fork
- Leading Strand - top strand of the fork, continuously synthesised (5’ –> 3’ direction)
- Lagging Strang - bottom strand of the fork, synthesised in short pieces called Okazaki Fragments which must be joined together (5’ –> 3’ direction)
What is an RNA primer?
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis.
It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA
What is the role of DNA primase?
RNA primes DNA for synthesis - to do this, a short RNA sequene is formed by DNA primase
Describe how the lagging strand is synthesied (step by step)
- DNA primase synthesises multiple short RNA fragment primers
- DNA polymerase adds to RNA primer (this begins the Okazaki fragment)
- A ribonuclease removes RNA primer (exonuclease)
- Repair DNA polymerase replaces RNA with DNA.
- DNA ligase joins the two fragments together (ATP).
What is proof reading?
Proof reading ensures that there are no replication errors (i.e. that the strands are replicated correctly)
What are the major differences between DNA and RNA?
- DNA - ds polynucleotide
- RNA - either ds or ss polynucleotide
- DNA - sugar = deoxyribose
- RNA - sugar = ribose
- DNA base = A, T, C, G
- RNA bases = A, U, C, G
What is a gene?
Gene = a unit of inheritance
Define transcription
The process in which nucleotide information in the DNA is copied into RNA is called transcription
DNA –> RNA (i.e. making an RNA copy of DNA)
What are the functional classes of RNA
- mRNA (messenger)
- tRNA (transfer)
- rRNA (ribosomal)
What is a transcripton factor?
Gene transcription involves special gene regulatory proteins called Transcription Factors
What is a promoter?
The “start” of a gene contains DNA sequences that are important in bringing about Transcription, they are the site at which the complex assembles called Gene promoters
Name and describe the function of the three (eukaryotic) RNA polymerases
- RNA Polymerase I -Transcribes rRNA genes
- RNA Polymerase III- Transcribes tRNA and 5S RNA genes
- RNA Polymerase II - Transcribes genes encoding proteins into mRNA
What is the basal transcription complex?
The BTC is a gene promotor than enables RNA Pol. II to be phosphorylated thereby enabling transcription to take place
What is the general role of the transcription factors?
TFs can interact with each other and the Basal Transcription Complex to modulate transcription.
They have specific DNA sequences which “bend DNA” on binding, i.e. cause the DNA to start to unwind
List the steps in transcritpion
- TFIID binds to TBP (at TATA box)
- TFII A and B bind
- RNAP II binds (to B which is bound to D)
- TFII E, H and J bind
what is pre-mRNA?
pre-mRNA or the primary transcript is the initial RNA produced during transcription (i.e. what needs to be processed into mRNA)
Define the following:
- Intron
- Exon
- Splicing
- Spliceosome
- Splice donor site
- Splce acceptor site
- intron - sequences in the gene which are transcribed but are edited out of the final mRNA
- exon - coding sequences (form part of the final mRNA)
- splcing - a type of pre-mRNA processing by which introns are removed (cut out)
- spliceosome - complex formed by the binding or the small ribonuclear proteins with pre-mRNA
- splice donor site - GU sequence at the 5’ end of the intron
- splice acceptor site - AG sequence at the 3’ end of the intron
Explain the steps involved in splicing?
- Splicing involes snRNPs
- U1 binds to the splice donor sequence
- Other snRNPs bind (U2 –> U6), it is U5 thyat binds to hte splice acceptor sequence
- Once all snRNPs have bound cleavage occurs (involves lariat formation between the A resiude of the intron)
- The lariat is removed and only the exon is left
What is polyadenylation?
Addition of the poly-A tail
This occurs one base at a time and is required for the creation of mature mRNA
What is the purpose of the 5’ cap?
the 5’ cap is added post-transcriptionally as a translational enhancer
it is fromed by the hydrolysis of the terminal triphosphate of mRNA (to diphosphate) - there is further modification - methylation (at N7)
Give an example of a splice mutation
Thalassaemia
Genetic, haematological disease - imablance in relative amounts of alpha and beta chains of haemaglobin caused by an inherrent issue in the splicing machinery
What is the typical structure of mRNA
5’ Cap – 5’ UTR – Coding Region – 3’ UTR – Poly-A Tail
In which direction is mRNA read?
5’ –> 3’
- mRNA reading begins at the 5’ cap*
- translation begins at the first AUG and then the ribosomes scans 5’ –> 3’ direction*
- (in triplets)*
Outline Translation
- mRNA is transcribed from DNA (pre-MRNA) and processed (this occurs in the nucleus)
- mRNA is transported out of the nucleus
- Translated in the 5’ –> 3’ direction into a protein (this occurs in the cytoplasm and on the rER)
What machinery is required for translation
Ribosomes - 2 subunits, proteins and rRNA
What is the function of tRNA (in translation)?
tRNA is the transporter of amino acids to the ribosome for translation. Each amino acid has a specific tRNA. tRNA code (triplet) is complementary to the mRNA triplet therefore it ensures fidelity of translation.
Outline the steps of initation of translation
- Step 1: dissociation of ribosome subunits (40S + 60S)
- Step 2: assembly of preinitiation complex (inc. initiation factors)
- Step 3: binding of mRNA to preinitiation complex,
- Step 4: binding of 60S subunit to the complex.
Outline the steps involved in elongation
- Step 1: binding of new tRNA carrying second amino acid to adjacent A site (amino acyl) site in frame with initiator Met (in P site)
- Step 2: catalysis of peptide bond between the two amino acids by peptidyl transferase on the 60s subunit
- Step 3: translocation of tRNA to P (peptidyl) site (original A site, which moves along) and dissociation of first tRNA.
What is an elongation factor?
Proteins that promote movement of ribosome along mRNA using GTP
Describe the steps of termination
- Step 1: recognition of stop codon causing release factors (proteins) bind to empty A site
- Step 2: release of peptide chain.
- Step 3: dissociation of release factors and ribosomes.
What is the role of aminoacyl tRNA
aminoacyl tRNA ensures the fidelity of the genetic code - there is one for each amino acid
How do antibiotics target replication & expression?
Certain antibiotics target and exploit the differences between eukaryotic and prokarytoic translation processes
Major differences can be found between the ribosomes and tranlation factors
Describe the key features of a nascent protein?
Newly synthesised proteins are very hydrophobic
(i.e. proteins that have just emerged from the ribosome)
Desribe the process involved in nascent proteins entering the secretory pathway
- Recognition of signal sequence by a protein-RNA complex: “Signal-Recognition Particle” (SRP)
- SRP binds to a receptor at the rER surface
- Translocation of the polypeptide into the lumen of rER
- Cleavage of signal sequence by signal peptidase (co-translational). Folding of the polypeptide then occurs.
List the potential post-translation modifications
- Disulphide bond formation (e.g. insulin)
- Proteolytic cleavage (e.g. insulinA and B chains)
- Addition of carbohydrate (Glycosylation)
- Addition of phosphate (Phosphorylation)
- Addition of lipid groups (Prenylation, Acylation)
- Hydroxylation (e.g. Collagen)
What is cell-based DNA cloning?
Construction of recombinant DNA molecules in vitro
Define: Restriction Endonucleases
Enzymes that cleave DNA at specific recognition sites, - produce “blunt” or “sticky” DNA ends
What is hybridisation?
A key method for detecting specific nucleic acid sequences in which homologous single-stranded DNA or RNA molecules combine via homologous base-pairing to form double-stranded molecules
Give some examples of hybridisation
- Southern blot hybridisation
- Northern blot hybridisation
- Colony blot hybridisation
- Chromosome in situ hybridisation
- Tissue in situ hybridisation
- Reverse hybridisation
What is hybridisation stringency
Hybridisation stringency (i.e. the power to distinguish between related sequences) increases with increase in temperature and decrease in salt concentration
Define PCR
In vitro method to allow selective amplification of a specific target DNA within a heterogeneous collection of DNA sequences (e.g. total genomic DNA or complex cDNA population).
What is a wobble pairing? What is its function?
Wobble rules - a wobble base pair is a pairing between two nucleotides in RNA molecules that does not follow Watson-Crick base pair rules. (i.e. G should only pair with C but can in fact also pair with U of RNA in position 3 of the codon). Function is to allow for broad specificity
