Topic 2/7 - Part 2 Flashcards
What are the three components of a nucleotide?
- a sugar, which has 5 carbon atoms, so is a pentose sugar
- a phosphate group, which is the acidic, negatively-charged part of nucleic acids
- a base that contains nitrogen and has either one or two rings of atoms in its structure
What is a nucleic acid? What are two examples of nucleic acids?
- nucleic acids are polymers (a large molecule composed of many repeated subunits) of nucleotides
- DNA and RNA are two nucleic acids
How are nucleotides linked together?
- covalent bonds are formed between the phosphate of one nucleotide and the pentose sugar of the next nucleotide, creating a strong backbone for the molecule of alternating sugar/photphate groups, with a base linked to each sugar
Differentiate between DNA and RNA
- The sugar within DNA is deoxyribose whilethe sugar is RNA is ribose; deoxyribose has one fewer oxygen atom than ribose
- There are usually two polymers of nucleotides in DNA but only one in RNA; polymers are often referred to as strands, so DNA is double-stranded while RNA is single-stranded
- In DNA, the nucleotide thymine is present while RNA has the nucleotide uracil instead
Describe the 3D structure of the DNA molecule
- DNA is a double helix made of two antiparallel strands of nucleotides linked by hydrogen bondings between complementary base pairs
- each strand consists of a chain of nucleotides linked by covalent bonds
- the two strands are parallel but run in opposite directions so they are said to be antiparallel; one strand is oriented in the direction 5’ to 3’ and the other is oriented in the direction 3’ to 5’
- the two strands are wound together to form a double helix
- the strands are held together by hydrogen bonds between the nitrogenous bases; A bonds with T and G bonds with C
List the four nitrogenous bases of DNA
- Adenine
- Cytosine
- Guanine
- Thymine
List the four nitrogenous bases of RNA
- Adenine
- Cytosine
- Guanine
- Uracil
What is meant by complementary base pairing?
- adenine is always paired with thymine (or uracil) and they therefore complement each other by forming base pairs
- guanine is always paired with cytosine and they therefore complement each other by forming base pairs
- purines pair up with pyrimidines
Pyrimidine
- cytosine, thymine, uracil
- one ring
Purine
- guanine, adenine
- two rings fused together
Double helix
- used to describe the strcture of DNA
- two strands that wind around each other like a twisted ladder.
- each strand has a backbone made of alternating groups of sugar (deoxyribose) and phosphate groups.
How did Crick and Watson discover the structure of DNA?
- The structural organisation of the DNA molecule was correctly proposed in 1953 by James Watson and Francis Crick
- These British scientists constructed models to quickly visualise and assess the viability of potential structures
- Their efforts were guided by an understanding of molecular distances and bond angles developed by Linus Pauling, and were based upon some key experimental discoveries:
1. DNA is composed of nucleotides made up of a sugar, phosphate and base
2. DNA is composed of an equal number of purines (A + G) and pyrimidines (C + T)
3. DNA is organised into a helical structure - Using trial and error, Watson and Crick were able to assemble a DNA model that demonstrated the following:
1. DNA strands are antiparallel and form a double helix
2. DNA strands pair via complementary base pairing (A = T ; C Ξ G)
3. Outer edges of bases remain exposed (allows access to replicative and transcriptional proteins) - As Watson and Crick’s model building was based on trial and error, a number of early models possessed faults:
1. The first model generated was a triple helix
2. Early models had bases on the outside and sugar-phosphate residues in the centre
3. Nitrogenous bases were not initially configured correctly and hence did not demonstrate complementarity
Explain what is meant by ‘semi-conservative’ in terms of DNA replication
- DNA replication is a semi-conservative process, because when a new double-stranded DNA molecule is formed, it contains one original strand and one newly synthesized strand
- the two strands of the double helix separate, each strand serving as a guide
- new strands are creating by adding nucleotides and linking them together
- because of complementary base pairing, the base sequence on the template strand determines the base sequence of the new strand
- the result is two DNA molecules, both composed of an original strand and a newly synthesized strand, and both identical to the original DNA molecule
Outline the three hypotheses that had been proposed for the replication of DNA
- Conservative Model – An entirely new molecule is synthesised from a DNA template
- Dispersive Model – New molecules are made of segments of new and old DNA
- Semi-Conservative Model – Each new molecule consists of one newly synthesised strand and one template strand (this is the currently accepted model)
How did Meselson and Stahl’s results support the theory of semi-conservative replication of DNA?
- Meselson and Stahl were able to experimentally test the validity of these three models using radioactive isotopes of nitrogen
- Nitrogen is a key component of DNA and can exist as a heavier 15N or a lighter 14N
- DNA molecules were prepared using the heavier 15N and then induced to replicate in the presence of the lighter 14N
- DNA samples were then separated via centrifugation to determine the composition of DNA in the replicated molecules
- The results after two divisions supported the semi-conservative model of DNA replication
- After one division, DNA molecules were found to contain a mix of 15N and 14N, disproving the conservative model
- After two divisions, some molecules of DNA were found to consist solely of 14N, disproving the dispersive model
Differentiate between the leading strand and the lagging strand
When replication begins, the two parent DNA strands are separated. One of these is called the leading strand, and it is replicated continuously in the 3’ to 5’ direction. The other strand is the lagging strand, and it is replicated discontinuously in short sections.
Outline the role of helicase
- Helicase unwinds and separates the double-stranded DNA by breaking the hydrogen bonds between base pairs
- This occurs at specific regions (origins of replication), creating a replication fork of two strands running in antiparallel directions
- The two separated polynucleotide strands will act as templates for the synthesis of new complementary strands
Outline the role of DNA gyrase
- DNA gyrase reduces the torsional strain created by the unwinding of DNA by helicase
- It does this by relaxing positive supercoils (via negative supercoiling) that would otherwise form during the unwinding of DNA
Outline the role of single stranded binding proteins
- SSB proteins bind to the DNA strands after they have been separated and prevent the strands from re-annealing
- These proteins also help to prevent the single stranded DNA from being digested by nucleases
- SSB proteins will be dislodged from the strand when a new complementary strand is synthesised by DNA polymerase III
Outline the role of DNA primase
- DNA primase generates a short RNA primer (~10–15 nucleotides) on each of the template strands
- The RNA primer provides an initiation point for DNA polymerase III, which can extend a nucleotide chain but not start one
Outline the role of DNA polymerase III
- Free nucleotides align opposite their complementary base partners (A = T ; G = C)
- DNA pol III attaches to the 3’-end of the primer and covalently joins the free nucleotides together in a 5’ → 3’ direction
- As DNA strands are antiparallel, DNA pol III moves in opposite directions on the two strands
- On the leading strand, DNA pol III is moving towards the replication fork and can synthesise continuously
- On the lagging strand, DNA pol III is moving away from the replication fork and synthesises in pieces (Okazaki fragments)
Outline the role of DNA polymerase I
- As the lagging strand is synthesised in a series of short fragments, it has multiple RNA primers along its length
- DNA pol I removes the RNA primers from the lagging strand and replaces them with DNA nucleotides
Define Okazaki fragment
Okazaki fragments are short, newly synthesized DNA fragments that are formed on the lagging template strand during DNA replication.
Outline the role of DNA ligase
- DNA ligase joins the Okazaki fragments together to form a continuous strand
- It does this by covalently joining the sugar-phosphate backbones together with a phosphodiester bond
Explain how PCR works and what it does
- polymerase chain reaction
- technique used to make many copies of a selected DNA sequence
- only a very small quantity of the DNA is needed at the start
Steps:
- DNA is loaded into the PCR machine
- PCR machine separates DNA strands by heating them up to 95 C for 15 seconds
- PCR machine quickly cools to 54 C
- A large amount of primers are present and they bind rapidly to the target sequences, preventing the re-annealing of the parent strands
- Copying of the single parent strands then starts from the primers
- Using the single strands with primers as templates, the synthesis of double-stranded DNA begins using the enzyme Taq DNA polymerase
- PCR machine heats up to 72 C, which is the optimum temperature for Taq DNA polymerase, for 80 seconds
- PCR machine heats up to 95 degrees again and begins the next cycle
Explain why Taq DNA polymerase is used in PCR
- adapted to be very heat-stable to resist denaturation
- can resist the brief 95 C used to separate the DNA strands