Nucleic Acids and Gene Expression Flashcards
Draw a nucleotide
What is the difference between a nucleotide and a nucleoside?
Nucleotide: base, sugar and phosphate group
Nucleoside: base and sugar only
What are the bases found in DNA and RNA?
Pyrimidines (small): Cytosine, Thymine (only in DNA), Uracil (only in RNA)
Purines (big): Guanine and Adenine
Explain the struture of a single DNA chain
A chain of deoxyribonucleotide units linked by phosphodiester bonds
3’ OH of sugar liked to phosphate linked to 5’ OH of next sugar
5’ and 3’ ends not symmetrical and conventionally written from 5’ to 3’
Explain the structure of a double-stranded DNA molecule
A & T: 2 H-bonds LESS STABLE
C & G: 3 H-bonds MORE STABLE
Chains are antiparallel
There are major (backbones are further away) and minor (backbones are closer together) grooves
Deoxyribose and phosphate groups = outside of helix (negative charges outside)
Bases = inwards and flat planes are perpendicular to the helix
How can a DNA molecule be melted and re-annealed?
MELTED: heat/ low salt - denatured
RE-ANNEALED: cool/ high salt - hybridise
Compare the genome of E. coli and Homo sapiens
- E. coli:* single circular DNA molecule; 4.6X106 base pairs; length= 1.4mm
- H. sapiens*: 3X109 base pairs divided into chromosomes each consisting of a single linear DNA molecule with 200X106 base pairs
Chromosomes only visible just before cell division
Explain the packagining of DNA
DNA tightly packaged to form a complex (chromatin) with proteins
Chromatin condenses to produce chromosomes
Lowest level of packaging = nucleosomes - DNA wrapped around histone proteins approx 200bp
Nucleosome: histone (+ve charge) DNA (-ve charge due to sugar-phosphate backbone)
8 histones- histone 1 between nucleosomes causes 7 fold condensing; further packaging causes 40 fold condensing
What is the human karyotype?
An organised profile of somone’s chromosomes
Humans have 46 chromosomes (44 autosomal and 2 sex)
XX= female
XY= male
Explain semi-conservative DNA replication
Each new daughter molecule of DNA has one new strand and one old strand from the parental DNA
Both strands of DNA are complementary and so each strand serves as a template for the synthesis of a new strand - generating 2 identical copies.
Explain the roles of DNA helicase and DNA polymerase
DNA helicase = uses ATP to break H-bonds between base pairs (unwinds the DNA helix)
DNA polymerase = adds nucleotides to 3’ end of a growing chain (synthesis of new DNA).
Requires a template strand, an olgionucleotide (short DNA sequence) primer and dNTPs (deoxynucleotide triphosphate)
Synthesis occurs 5’ to 3’; energy released by hydrolysis of triphosphate; free 3’ hydroxyl group is needed.
How are nucleoside analogs used as drugs?
Consists of: nucleic acid analog (structurally similar to a nucleic acid) and a sugar
DO NOT have a free 3’ hydroxyl group and so can terminate synthesis of a new DNA molecule
examples include;
dideoxycytidine (ddC) = HIV
azidothyminide (AZT) = HIV
acyclovir = herpes
cytosine arabinose = chemotherapy
What is the leading strand?
One which has its 3’ end closest to the replication fork
DNA synthesis is continuous as the 3’ end is moving in the same direction as the replication fork
What is the lagging strand?
Has its 3’ end at the opposite side to the replication fork
DNA synthesis is discontinuous and occurs using small Okazaki fragments
What is an Okazaki fragment?
Short sequences of DNA which are synthesised from the lagging strand
What is the replication fork?
The specific point in a DNA molecule where DNA synthesis is occuring.
Explain how the leading strand is synthesised
- Synthesis always occurs from 5’ to 3’ = CONTINUOUS
- A RNA primer (synthesised by DNA primase) is needed to start replication at the replication origin
- DNA polymerase adds dNTPs to the 3’ end to extended the molecule
Explain how the lagging strand is synthesised
- Synthesis is discontinuous
- A RNA primer is needed (synthesised by primase)
- DNA polymerase adds dNTPs to the RNA primer - producing a new Okazaki fragment
- The old RNA primer is removed by a ribonuclease via exonuclease activity
- DNA polymerase synthesises DNA to replace the RNA primer region
- DNA ligase then joins the Okazaki fragments using ATP
What other proteins are involved at the site of the replication fork?
SINGLE STRAND DNA BINDING PROTIEN
Prevents the folding of single stranded DNA
SLIDING CLAMP
Ensures that DNA polymerase remains in the correct location as the lagging strand may loop preventing synthesis
How is accuracy maintained in DNA replication?
Errors can occur approx. 1 in about 109 bp; can potentially be dangerous
Before adding a new nucleotide, the previous is checked for the correct base-pairing by DNA polymerase
Incorrect base = removed by 3’ to 5’ exonuclease activity (of DNA polymerase) and the correct nucleotide is added
Inaccurate RNA primers are also replaced by accurate DNA
How does E. coli replicate?
There is only one replication origin, OriC, resulting in 2 replication forks proceeding simultaneously in opposite directions - meet at the other side of the circle
How do eukaryotic chromosomes replicate?
Due to the size of the chromosomes there are multiple replication origins
Thus giving bi-directional replication forks
Replication finishes once the forks have met
What are the stages of the cell cycle?
INTERPHASE
G1 = gap phase 1; before DNA synthesis = 10hrs
G0 = cells have stopped divinding
S = synthesis; DNA replicates = 9hrs
G2 = gap phase 2; between DNA synthesis and mitosis, sister chromatids seen = 4hrs
MITOSIS = cell division = 1hr
How do chromosomes segregate at metaphase?
Chromosomes are condensed and attach to the spindle on the central plane
During anaphase they are seprated to opposite spindle poles
Explain the major differences between DNA and RNA
DNA:
- hereditary material
- organised into genes (units of inheritance)
- double stranded
- deoxyribose sugar
RNA:
- initial product of gene expression
- single stranded
- ribose sugar
- major species; tRNA, rRNA, mRNA
Define transcription
DNA ►RNA
The process in which nucleotide information in the DNA is copied into RNA
What are the major types of RNA?
tRNA = transfer RNA; transfers an amino acid to a complementary codon on mRNA
rRNA = ribosomal RNA; the major component of ribosomes
mRNA = messenger RNA; transfers genetic information from within the nucleus to the cytoplasm
What are the major classes of RNA polymerase?
RNA Polymerase I = transcribes rRNA genes
RNA Polymerase III = transcribes tRNA and 5S rRNA genes
RNA Polymerase II = transcribes genes encoding protiens into mRNA
What is a gene promoter?
The DNA sequence at which the initiation complex assembles (RNA Pol can bind) is a gene promoter
e.g. for the initiation of transcription by RNA Pol II the gene promoter is TATA repeated
What is a transcription factor?
A protein which can bind to DNA to regulate the amount of transcription a gene undergoes
Usually found just before a gene promoter (e.g. TATA)
Can either be
- transcriptional activators - activate gene expression
- transcriptional repressor - suppress gene expression
What are the two main proteins involved in transcription?
RNA Polymerase (enz.) = carries out gene transcription
Transcription Factors = regulatory proteins
Give a brief overview of the process of transcription
- DNA strand unwinds
- Ribonucleotides base pair with DNA bases on one strand
- Bases join via phosphodiester bonds; thus the RNA chain grows one base at a time going from 5’ to 3’ (sense strand)
CHECKKKK
What is the basal transcription complex?
A collection of proteins which allow RNA Pol II to be phosphorylated and take part in transcription
Transcription factors must also bind; otherwise a basal (low) level of transcription occurs
Describe the formation of the Basal Transcription Complex
- TF IID binds to TATA
- causes DNA helix to partially unwind, widening the minor groove allowing for more contact with bases
- Both TF IIA and TF IIB bind
- TF IIB binds to both TF IID and RNA Pol II
- RNA Pol II binds along with TF IIF
- TF IIJ, TF IIE, TF IIH all bind to RNA Pol II
- TF IIH promotes further unwinding and allows phosphorylation of RNA Pol II so that it is activated
- Transcription Factors also bind causing DNA to bend and interact to regulate transciption
What is the mechanism behind transcription factors?
Transcription factors can “bend DNA” as they bind thus casuing interactions between other TFs and also the Basal Transcription Complex to regulate transcription
Explain the events that take place in pre-mRNA processing
Inital RNA produced from a gene = pre-mRNA/ heterogenous nuclear RNA (hn RNA)
Must be processed in the nucleus so can be used as mRNA for translation in the cytoplasm
- Gene promoter (at 5’ end); segments forming final RNA = exons; sequences transcribed but edited out of the final mRNA = introns
- Adding a 5’ cap
- Adding a poly- A tail
What is a splice donor site?
The base sequence: AGGU
At the end of an exon and the start of the intron
Allows the binding of U1 which is a small nuclear ribonucleoprotein (snRNP)
What is a splice acceptor site?
Pyr15NCAG
Pyr = Pyrimidine C/U
N = any base
Allows U5 (a snRNP) to bind to it
Explain how mRNA splicing occurs.
- Binding of snRNPs; U1 to splice donor site, U2, U4, U6 to intron and U5 to splice acceptor site
- Once all are bound, the spliceosome is formed, cleaving the splice donor sequence i.e. GU detaches
- Attaches to an A on the intron via a phosphodiester bond
- Bonding is unique, 5’ Pi of G to 2’ OH of A
- Cleavage of AG (end of intron)
- Formation of a lariat structure which is unique to RNA and in then destroyed
- Adjacent exons can then ligate
What is a spliceosome?
The formation of the splicing complex which involves snRNPs (U1, U2, U4, U6 and U5)
Explain how the “cap” is added to pre-mRNA
The cap can protect mRNA at the 5’ end to enhance translation of mRNA
- Hydrolysis of the triphosphate on 5’ end to a diphosphate
- Can react with the alpha-phosphate on GTP to produce a 5’-5’ phosphate linkage
- Further modification occurs to form a 7-methlguanylate cap; methylation of the purine ring at N7
Explain how the poly- A tail is addedd to pre-mRNA
Via a process called polyadenylation
A is added one at a time till roughly a tail of 200 bases is formed
Tail is downstream of the sequence AAUAAA
How do mutations in splice sites cause human disease?
Example => Thalassemia (imbalance of alpha-chains and beta-chains making up Haemoglobin)
Mutation in splice site causes a change in intron/exon length and so the desired/ functional mRNA strand is not produced
How is the complexity of a genome determined?
genome = the complete DNA sequence of an organism
The complexity of an organism is not determined by the genome size (C-value paradox)
What are small non-coding RNAs (ncRNAs)?
Some RNA sequences are not translated into protein
- House-keeping ncRNA: rRNA, tRNA, snRNA (splicosome)
- Regulatory ncRNA:
- microRNA = controls the transcription of genes
- RNAi (RNA interference) = targetted inhibition of genes used in viral defence (uses miRNA and siRNA - micro and small interfering)
- piRNA (piwi-interacting RNA) = involvement in epigenetics in particular gene-silencing in germ line cells
- long ncRNA = many roles including regulating gene transcription, protein translation and X-chromosome activation
What are the biochemical mechanisms underlying gene regulation?
- DICER ( an endonuclease which cleaves double-stranded RNA dsRNA) digests dsRNA into small fragments approx. 20bp forms siRNA
- One strand is removed from siRNA via Agronaute and Piwi proteins
- Forms an anti-sense strand which binds to mRNA forming a siRNA-mRNA complex
- mRNA is then cleaved by the RISK complex to form two segements and so can not be translated = SILENCED
What is the role of micro RNAs (miRNAs)?
miRNA = function in the transcriptional and post-transcriptional regulation of gene expression. Inhibit target mRNA = base pairing with incomplete complementarity
How are micro RNAs produced?
Produced in the nucleus initially as larger pri-miRNAs
- Transcribed by either RNA polymerase II or III to form pri-miRNA
- Shortened via RNase III endonuclease (Drosha) and DGCR8
- Forms pre-miRNA which is transported to the cytoplasm
- Further cleaving occurs via Dicer, Argonaute and TRBP (RNA Binding Protein)
- RISC then forms a single stranded miRNA strand
How can miRNA be involved in human disease?
Part of chromosome 14 can be deleted and so leads to the loss of miRNA and promotes Chronic Lymphoid Leukaemia
Supplementing miRNA in mice with CLL results in treatment of the cancer; miRNA = theraputic
What are the STOP codons
UAA
UAG
UGA
What is the START codon?
AUG (methionine)
How is translation initiated?
- The ribosome subunits dissociate into 40S and 60S (eukaryotes)
- The pre-initiation complex assembles
- met-tRNA (will bind to START codon)
- initiation factors
- 40S subunit
- mRNA binds to pre-initiation complex
- The 60S subunit binds (GTP => GDP +Pi)
How does elongation occur in translation?
- Second tRNA molecule binds, carrying second amino acid to amino acyl (A) site
- Peptide bond forms between the two amino acids, catalysed by peptidyl transferase on 60S
- First tRNA dissociates
- Second tRNA enters the peptidyl (P) site
Elongation Factors = proteins which promote the movement of the ribosome along mRNA (uses GTP)
How is translation terminated?
- STOP codon is recognised (UAA, UGA, UAG) (tRNA doesnt bind, release factor does)
- The peptide chain is released via peptidyl transferase transfering the protein chain to water
- Dissociation of release factors and ribosomes
What is the role of aminoacyl tRNAs?
Aminoacyl tRNA synthetase (enz.) = specific for each amino acid
Ensures the correct amino acid is selected
AA => adenylated AA (AMP and enz. attached) => AA (after binding to tRNA; AMP and enz. dissociates, AA attached to 3’OH of tRNA)
Why do some antibiotics only inhibit protein synthesis in prokaryotes and not eukaryotes?
Antibiotics selectively inhibit prokaryotes due to differences between prokaryotic and eukaryotic ribosomes and translation factors.
Examples
- streptomycin = inhibits initiation
- tetracycline = inhibits AA-tRNA binding
- erythromycin = inhibits translocation
What are the features of a newly synthesised protein so that it can enter the secretory pathway?
SIGNAL SEQUENCE = enriched in hydrophobic amino acids (Leu, Ile, Phe, Trp, Tyr, Ala)
- The signal sequence is recognised by the protein-RNA complex = Signal-Recognition Particle (SRP) stopping translation
- The SRP binds to a receptor on the RER surface, resuming translation
- The growing peptide is translocated to the lumen of the RER
- The signal sequence is cleaved by signal peptidase and so folding occurs
What are the methods by which new proteins can by modified post-translationally?
Increases diversity and makes proteins fully functional
- Disulphide bond formation
- Proteolytic cleavage
- Addition of
- carbohydrate (glycosylation)
- phosphate (phosphorylation)
- lipids (prenylation/acylation)
- Hydroxylation
What is hybridisation?
Joining of a nucleic acid probe (usually labelled for identification) and a specific sequence of DNA -single stranded- or RNA so that desired sequences can be detected.
- Target DNA = immobilised on a solid support (nylon/nitrocellulose membrane)
- Probe DNA = in a solution (labelled either radioactively or fluorescently)
What are the different types of hybridisation?
What is hybridisation stringency?
The power to distinguish between related sequences; increases with
- increase in temperature
- decrease in Na+ concentration
DENATURES DNA
What factors affect the amount of energy needed to denature a DNA probe?
- Strand length; longer = more H-bonds to break
- Base compositon; G-C has one more H-bond so harder to break
- Chemical environment;
- Na+ = stabilise DNA (neutralises charge on phosphate backbone)
- denaturants e.g. urea/formamide = destabilise DNA
What is the difference between high and low stringency?
HIGH = duplex only forms with one-to-one complementarity
LOW = some mismatch may occur between bases and annealing still occurs
At what temperature is hybridisation carried out in mammals?
Melting temperature = midpoint for transition from double strand to single strand
25C below melting temperature
mammals = 87C
What is PCR?
PCR = Polymerase Chain Reaction
in vitro method which allows a specific DNA sequence to be amplified within a heterogeneous collection
How is PCR carried out?
- Two primers are produced which are complimentary to the target DNA - some sequence information is needed
- Annealing occurs between primers and heat-denatured DNA (low temperature)
- Taq (thermostable) DNA polymerase along with dNTPs extend the primer to synthesis new strands
- The DNA molecule can be denatured and the cycle repeated to increase the number of target DNA molecules
- Denature = 94C
- Anneal = 50-60C
- Extend = 72C
What is PCR used for?
- Typing genetic markers
- Detecting point mutations
- cDNA (a copy of mRNA) cloning
- Genome walking
- Gene expression (mRNA => DNA)
- Introducing mutations experimentally
- DNA sequencing
- DNA microarrays
How are primers designed for PCR?
- Approx. 20 nucleotides long - provides specificity for target sequence
- Avoid tandem repeats as hairpins may form
- Equal melting temperature (same % of G-C and length)
- Avoid complemtarity of bases at 3’ end (primer dimers may form)
What are restriction enzymes/endonucleases?
Type II restriction endonucleases can cleave DNA at specific recognition sites (usually 4-8bp palindromic sequences)
May produce a blunt or “sticky” (overhang) end
Longer recogniton site- less frequent cleaving
Type of primitive immune system; host DNA is protected due to methylation at the recognition site and so only cleaves unmethylated DNA from invading organisms
