Nucleic Acids

Question 1

Describe the structre of DNA

Answer 1

Phosphates (and sugars) form an outer backbone and nitrogenous bases are packaged within the interior

DNA is composed of an equal number of purines (A + G) and pyrimidines (C + T)

Nitrogenous bases are paired (purine + pyrimidine) within the double helix

Two strands must run in antiparallel directions

Adenine and thymine paired via two hydrogen bonds, whereas guanine and cytosine paired via three hydrogen bond

Question 2

Describe DNA Repllication

Answer 2

Helicase

Helicase unwinds and separates the double-stranded DNA by breaking the hydrogen bonds between base pairs creating a replication fork of two strands running in antiparallel directions

DNA Gyrase

DNA gyrase reduces the torsional strain created by the unwinding of DNA by helicase

Single Stranded Binding (SSB) Proteins

SSB proteins bind to the DNA strands after they have been separated and prevent the strands from re-annealing

DNA Primase

DNA primase generates a short RNA primer 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

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)

DNA Polymerase I

DNA pol I removes the RNA primers and replaces them with DNA nucleotides

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

Question 3

Where does DNA polymerase III obtain energy for replication?

Answer 3

Free nucleotides exist as deoxynucleoside triphosphates (dNTPs) – they have 3 phosphate groups
DNA polymerase cleaves the two additional phosphates and uses the energy released to form a phosphodiester bond with the 3’ end of a nucleotide chain

Question 4

What are the functions of non-coding DNA?

Answer 4

Satellite DNA - Short tandem repeats for DNA Profiling / Structural component of centromeres

Telomeres - Protects against chromosonal deterioration durring replication

Introns - Removed by RNA splicing

Non - Coding RNA genes - Molecules not translated for protein 9 ex. tRNA)

Gene regulatory sequences - Involved in transcription (Promoters etc)

STING

Question 5

With reference to nucleosomes explain how DNA is packaged

Answer 5

The DNA is complexed with eight histone proteins (an octamer) to form a complex called a nucleosome

Nucleosomes are linked by an additional histone protein (H1 histone) to form a string of chromatosomes

These then coil to form a solenoid structure (~6 chromatosomes per turn) which is condensed to form a 30 nm fibre

These fibres then form loops, which are compressed and folded around a protein scaffold to form chromatin

Chromatin will then supercoil during cell division to form chromosomes that are visible (when stained) under microscope

Question 6

Define nucleosome

Answer 6

A nucleosome consists of a molecule of DNA wrapped around a core of eight histone proteins (an octamer)

Question 7

Explain the role of the Promoter Region in Transcription

Answer 7

Promoter

The non-coding sequence responsible for the initiation of transcription

The core promoter is typically located immediately upstream of the gene’s coding sequence

The promoter functions as a binding site for RNA polymerase (the enzyme responsible for transcription)

The binding of RNA polymerase to the promoter is mediated and controlled by an array of transcription factors in eukaryotes

These transcription factors bind to either proximal control elements (near the promoter) or distal control elements (at a distance)

Question 8

What is the coding sequance in Transcription

Answer 8

The region of DNA that is transcribed by RNA polymerase is called the coding sequence

Question 9

What is the terminator sequance in Transcription

Answer 9

RNA polymerase will continue to transcribe the DNA until it reaches a terminator sequence

Question 10

Distiguish between sense and antisense strands

Answer 10

The antisense strand is the strand that is transcribed into RNA
Its sequence is complementary to the RNA sequence and will be the “DNA version” of the tRNA anticodon sequence
The antisense strand is also referred to as the template strand

The sense strand is the strand that is not transcribed into RNA
Its sequence will be the “DNA version” of the RNA sequence (i.e. identical except for T instead of U)
The sense strand is also referred to as the coding strand (because it is a DNA copy of the RNA sequence)

Question 11

Distinguish between the template strand and coding strands

Answer 11

The Template strand is the strand that is transcribed into RNA
Its sequence is complementary to the RNA sequence and will be the “DNA version” of the tRNA anticodon sequence
The antisense strand is also referred to as the template strand

The Coding strand is the strand that is not transcribed into RNA
Its sequence will be the “DNA version” of the RNA sequence (i.e. identical except for T instead of U)
The sense strand is also referred to as the coding strand (because it is a DNA copy of the RNA sequence)

Question 12

Outline transcription

Answer 12

The process of transcription can be divided into three main steps: initiation, elongation and termination

In initiation, RNA polymerase binds to the promoter and causes the unwinding and separating of the DNA strands
Elongation occurs as the RNA polymerase moves along the coding sequence, synthesising RNA in a 5’ → 3’ direction
When RNA polymerase reaches the terminator, both the enzyme and nascent RNA strand detach and the DNA rewinds

Question 13

How is mRNA modified after transcription

Answer 13

Capping

Capping involves the addition of a methyl group to the 5’-end of the transcribed RNA
The methylated cap provides protection against degradation by exonucleases
It also allows the transcript to be recognised by the cell’s translational machinery (e.g. nuclear export proteins and ribosome)

Polyadenylation

Polyadenylation describes the addition of a long chain of adenine nucleotides (a poly-A tail) to the 3’-end of the transcript
The poly-A tail improves the stability of the RNA transcript and facilitates its export from the nucleus

Splicing

Within eukaryotic genes are non-coding sequences called introns, which must be removed prior to forming mature mRNA
The coding regions are called exons and these are fused together when introns are removed to form a continuous sequence

Question 14

How does splicing allow for a larger no. of combinations?

Answer 14

Splicing can also result in the removal of exons – a process known as alternative splicing

The selective removal of specific exons will result in the formation of different polypeptides from a single gene sequence

Question 15

How is gene expression regulated?

Answer 15

Transcription factors form a complex with RNA polymerase at the promoter

RNA polymerase cannot initiate transcription without these factors and hence their levels regulate gene expression

Activator proteins bind to enhancer sites and increase the rate of transcription (by mediating complex formation)
Repressor proteins bind to silencer sequences and decrease the rate of transcription (by preventing complex formation)

Question 16

How do environmental factors affect gene expression?

Answer 16

Chemical signals within the cell can trigger changes in levels of regulatory proteins or transcription factors in response to stimuli

This allows gene expression to change in response to alterations in intracellular and extracellular conditions

Ex. Red and Blue flowers depending on pH

Question 17

How is transcription regulated in eukaryotes?

Answer 17

Typically the histone tails have a positive charge and hence associate tightly with the negatively charged DNA

-Adding an acetyl group to the tail (acetylation) neutralises the charge, making DNA less tightly coiled and increasing transcription
-Adding a methyl group to the tail (methylation) maintains the positive charge, making DNA more coiled and reducing transcription

Question 18

State the different types of chromatin

Answer 18

When DNA is supercoiled and not accessible for transcription, it exists as condensed heterochromatin

When the DNA is loosely packed and therefore accessible to the transcription machinery, it exists as euchromatin

Question 19

What is the correlation between methylation patterns and transcription?

Answer 19

Increased methylation of DNA decreases gene expression (by preventing the binding of transcription factors)
Consequently, genes that are not transcribed tend to exhibit more DNA methylation than genes that are actively transcribed

Question 20

What are the types of RNA? (EXTRA)

Answer 20

here are three main types of RNA which cooperate to complete this goal:

Messenger RNA (mRNA) – a transcript copy of a gene which encodes a specific polypeptide
Transfer RNA (tRNA) – carries the polypeptide subunits (amino acids) to the organelle responsible for synthesis (ribosome)
Ribosomal RNA (rRNA) – a primary component of the ribosome and is responsible for its catalytic activity

Additionally, cells may produce other variants of non-coding RNA to support and regulate the expression of genes:

Small nuclear RNA (snRNA) – a component of the spliceosome (involved in splicing of introns)
Short interfering RNA (siRNA) – moderates gene expression levels via RNA interference (RNAi)

Question 21

Describe the structure of a Ribosome

Answer 21

They consist of a large and small subunit:
The small subunit contains an mRNA binding site
The large subunit contains three tRNA binding sites – an aminoacyl (A) site, a peptidyl (P) site and an exit (E) site

Question 22

Describe the structure of tRNA

Answer 22

tRNA molecules fold into a cloverleaf structure with four key regions:

The acceptor stem (3’-CCA) carries an amino acid
The anticodon associates with the mRNA codon (via complementary base pairing)
The T arm associates with the ribosome (via the E, P and A binding sites)
The D arm associates with the tRNA activating enzyme (responsible for adding the amino acid to the acceptor stem)

Question 23

How does tRNA bind with an amino acid

Answer 23

Each tRNA molecule binds with a specific amino acid in the cytoplasm in a reaction catalysed by a tRNA-activating enzyme

Each amino acid is recognised by a specific enzyme (the enzyme may recognise multiple tRNA molecules due to degeneracy)

The enzyme binds ATP to the amino acid to form an amino acid–AMP complex linked by a high energy bond (PP released)
The amino acid is then coupled to tRNA and the AMP is released – the tRNA molecule is now “charged” and ready for use

The function of the ATP (phosphorylation) is to create a high energy bond that is transferred to the tRNA molecule

This stored energy will provide the majority of the energy required for peptide bond formation during translation

Question 24

Describe translation

Answer 24

Initiation

The first stage of translation involves the assembly of the three components that carry out the process (mRNA, tRNA, ribosome)

The small ribosomal subunit binds to the 5’-end of the mRNA and moves along it until it reaches the start codon (AUG)
Next, the appropriate tRNA molecule bind to the codon via its anticodon (according to complementary base pairing)
Finally, the large ribosomal subunit aligns itself to the tRNA molecule at the P site and forms a complex with the small subunit

Elongation

A second tRNA molecule pairs with the next codon in the ribosomal A site
The amino acid in the P site is covalently attached via a peptide bond (condensation reaction) to the amino acid in the A site
The tRNA in the P site is now deacylated (no amino acid), while the tRNA in the A site carries the peptide chain

Translocation

The ribosome moves along the mRNA strand by one codon position (in a 5’ → 3’ direction)
The deacylated tRNA moves into the E site and is released, while the tRNA carrying the peptide chain moves to the P site
Another tRNA molecules attaches to the next codon in the now unoccupied A site and the process is repeated

Termination

The final stage of translation involves the disassembly of the components and the release of a polypeptide chain

Elongation and translocation continue in a repeating cycle until the ribosome reaches a stop codon
These codons do not recruit a tRNA molecule, but instead recruit a release factor that signals for translation to stop
The polypeptide is released and the ribosome disassembles back into its two independent subunits

Question 25

How does Translaation differ in prokaryotes?

Answer 25

Prokaryotes lack compartmentalised structures (like the nucleus) and so transcription and translation need not be separated

Ribosomes may begin translating the mRNA molecule while it is still being transcribed from the DNA template
This is possible because both transcription and translation occur in a 5’ → 3’ direction

Question 26

What is a polysome

Answer 26

A polysome (or a polyribosome) is a group of two or more ribosomes translating an mRNA sequence simultaneously

Question 27

What decides if a protein is intracellular or extracellular

Answer 27

If the protein is targeted for intracellular use within the cytosol, the ribosome remains free and unattached

If the protein is targeted for secretion, membrane fixation or use in lysosomes, the ribosome becomes bound to the ER

Protein destination is determined by the presence or absence of an initial signal sequence on a nascent polypeptide chain

-The presence of this signal sequence results in the recruitment of a signal recognition particle (SRP), which halts translation
-The SRP-ribosome complex then docks at a receptor located on the ER membrane (forming rough ER)
-Translation is re-initiated and the polypeptide chain continues to grow via a transport channel into the lumen of the ER
-The synthesised protein will then be transported via a vesicle to the Golgi complex (for secretion) or the lysosome
-The signal sequence is cleaved and the SRP recycled once the polypeptide is completely synthesised within the ER

Question 28

Describe primary structure of a protein

Answer 28

The first level of structural organisation in a protein is the order / sequence of amino acids which comprise the polypeptide chain
The primary structure is formed by covalent peptide bonds between the amine and carboxyl groups of adjacent amino acids

Question 29

Describe secondary structure of a protein

Answer 29

The secondary structure is the way a polypeptide folds in a repeating arrangement to form α-helices and β-pleated sheets
This folding is a result of hydrogen bonding between the amine and carboxyl groups of non-adjacent amino acids

Secondary structure provides the polypeptide chain with a level of mechanical stability (due to the presence of hydrogen bonds

Question 30

Describe tertiary structure of a protein

Answer 30

The tertiary structure is the way the polypeptide chain coils and turns to form a complex molecular shape (i.e. the 3D shape)
It is caused by interactions between R groups; including H-bonds, disulfide bridges, ionic bonds and hydrophobic interactions

Tertiary structure may be important for the function of the protein (e.g. specificity of active site in enzymes)

Question 31

Distinguish between fiberous and globular proteins

Answer 31

Fiberous / Globular
Shape - Long and Narrow / Spherical
Purpose- Structural / Functional
Sensitive - More / Less
Solubility - Insoluble / Soluble