Nucleic Acids Flashcards
Describe the structre of DNA
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
Describe DNA Repllication
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
Where does DNA polymerase III obtain energy for replication?
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
What are the functions of non-coding DNA?
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
With reference to nucleosomes explain how DNA is packaged
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
Define nucleosome
A nucleosome consists of a molecule of DNA wrapped around a core of eight histone proteins (an octamer)
Explain the role of the Promoter Region in Transcription
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)
What is the coding sequance in Transcription
The region of DNA that is transcribed by RNA polymerase is called the coding sequence
What is the terminator sequance in Transcription
RNA polymerase will continue to transcribe the DNA until it reaches a terminator sequence
Distiguish between sense and antisense strands
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)
Distinguish between the template strand and coding strands
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)
Outline transcription
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
How is mRNA modified after transcription
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
How does splicing allow for a larger no. of combinations?
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
How is gene expression regulated?
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)
How do environmental factors affect gene expression?
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
How is transcription regulated in eukaryotes?
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
State the different types of chromatin
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
What is the correlation between methylation patterns and transcription?
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
What are the types of RNA? (EXTRA)
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)
Describe the structure of a Ribosome
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
Describe the structure of tRNA
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)
How does tRNA bind with an amino acid
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
Describe translation
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
