DNA & Genomics Flashcards
Sugar-phosphate backbone shows directionality. What is meant by the 5’ end and 3’ end of the chain?
5’: free phosphate group attached to C5 of deoxyribose
3’: free hydroxyl group attached to C3 of deoxyribise
Compare the structure of DNA and RNA.
- Location
- Amount
- Macro features
- DS vs SS
- Chemical stability
- Size
- directionality (S)
- Sugar phosphate backbone (S) - Variation/forms
- Nt
- basic unit
- sugar residue (S/D)
- nitrogenous bases
- ratio of bases
- bond (S/D)
What are the advantages of DNA being double stranded?
- DNA mlcs more stable in structure as collective H bonds between base pairs strengthen double-helical structure
- One strand can act as a template for DNA repair of the other strand
- DNA can be replicated accurately via semi-conservative replication (each strand serves as a template for synthesis of a new complementary strand)
What is the importance of Chargaff’s rule/1:1 ratio in the structure of DNA?
⇒ indicate cbp, where A pairs w T, G pairs w C
⇒ H bonds between complementary bases stabilize struc of DNA
⇒ pairing between purine & pyrimidine→ constant width of 2.0nm between 2 sugar-phosphate backbone
What is the role of DNA?
Store info, pass it from one generation to the next
What makes DNA a suitable store of information?
- Can be accurately replicated: daughter cells have identical copies of DNA as parent cell
- Weak H bonds between 2 strands→ can separate & act as templates for new strand synthesis
- Complementary base pairing: A=T, G≡C - Stable molecule→ passed on to next gen wout loss of coded info
- Collectively, numerous H bonds hold 2 DNA strands tgt
- adj nucleotides in each strand joined by strong covalent phosphodiester bond - Backup of the code
- DNA is ds
- One strand can serve as template for the repair of the other, when mutations occur on either one - Coded info readily utilised/accessed
- Weak H bonds between 2 strands→ template strand can separate from non-template strand allowing transcription to take place
- Complementary base pairing: faithful transfer of info from DNA to RNA during transcription, which will subsequently be translated to protein
Describe the role of mRNA in protein synthesis.
- synthesized from transcription, by cbp w DNA serving as a template, where A=U, T=A, G w C
- Acts as a messenger and carrier of genetic code, to carry transcripts of info from gene in nucleus to ribosomes in cytoplasm/RER, where translation takes place, via nuclear pores
- template for translation
- each codon in the coding region specifies an aa in a polypeptide chain→ seq of codons on mRNA code for aa seq in a single polypeptide
- During translation:
> codons in mRNA cbp with anticodons of a specific tRNA carrying a specific aa
> mRNA has recognition sites that allow it to bind to the small ribosomal subunit
> mRNA has start/stop codons - gene expression can be regulated (varying the rate of mRNA synthesis/rate of breakdown)
Describe the role of tRNA in protein synthesis.
Brings in specific aa (to the growing polypeptide at ribosome) in a seq corresponding to seq of codons in mRNA.
It can facilitate translation because:
- 3’ end w CCA stem→ ability of tRNA w specific anticodon to bind to specific single aa during aa activation
- ability of its specific anticodon to base-pair to a specific mRNA codon during translation (A bp w U; G bp w C, by forming H bonds)→ ensures seq of nt on mRNA translated into a specific seq of aa in polypeptide chain.
tRNA is then released after aa joins polypeptide, and reused, attaching to another specific aa
Describe the role of rRNA in protein synthesis.
- associates w ribosomal proteins→ ribosomes
- small subunit: cbp between rRNA in mRNA binding site and mRNA, where A base pairs w U, G base pairs w C→ small subunit binds to mRNA
- large subunit
- enables binding of aminoacyl-tRNAs to Peptidyl site (P site) & Aminoacyl site (A site) via cbp
- part of it acts as peptidyl transferase, catalysing formation of peptide bond between 2 aa
What is the function of ribosomes?
Synthesise polypeptide under direction of mRNA by:
- hold tRNA and mRNA closely, allow interaction between codon of mRNA & anticodon of tRNA
- positions new aa for addition to growing polypeptide
- peptidyl transferase catalyses formation of peptide bonds between 2 aa
What’s the difference between ribosomes in prokaryotes and eukaryotes?
- eu: 80S (small (40S) + large (60S))
- pro: 70S (small (30S) + large (50S))
Define gene.
a specific seq of nucleotides in a DNA molecule, which codes for a specific seq of aa in one polypeptide chain. It’s located in a fixed position (locus) on chromosome/DNA mlc, and specifies a particular biological function (phenotype).
Template strand/antisense strand = non-coding seq, mRNA complementary to it. True/False?
True
Outline the features of the genetic code (+ definition)
Describes the manner in which a particular nucleotide sequence is translated into its corresponding amino acid sequence
- Triplet code, where each seq of 3 consecutive nt/codon codes for 1 aa
- If 1 nt coded for 1 aa→ only be 4 aa; If 2 nt code for 1 aa→ only be 16 (42) aa; If 3 nt code for 1 aa→ only be 64 (43) aa, > enough to code for all 20aa - Universal: same triplet of nt codes for same aa in all organisms (basis of genetic engineering)
- Degenerate: For some aa out of the 20, an aa may be coded for by >1 codon
- Non-overlapping: Codons read as successive groups of 3 nucleotides
- Continuous: no nucleotides ‘skipped’ between codons; code read as continuous seq of nt bases
- Includes ‘start’ & ‘stop’ seq
- Start codon: AUG→ signals initiation site for translation of mRNA into seq of aa. It codes for methionine
- Stop codon: UAA/UAG/UGA→ don’t code for any aa; stop signals for termination of polypeptide chain synthesis during translation
What is conservative DNA replication?
2 parental strands separate, act as templates for synthesis of new strands→ reassociate→ restored original double helix + daughter DNA molecule consisting of 2 newly synthesised strands
What is dispersive DNA replication?
parental DNA fragmented and dispersed→ daughter molecules w mixture of old & newly synthesised parts
Describe semi-conservative DNA replication.
parental DNA molecule separates into 2 single strands through breakage of H bonds, each act as template for synthesis of complementary daughter/new strand through cbp→ 2 new DNA molecule, each a hybrid of 1 parental strand & 1 newly synthesised strand
What is the advantage of having multiple Ori?
DNA polymerase can add dNTPs at a certain max rate (fixed). Multiple Ori in a single DNA molecule→ many DNA pol can work simultaneously→ speed up copying of very long DNA mlcs
Describe DNA replication: START
- replication starts at Ori
- Helicase breaks H bonds between complementary base pairs of 2 strands→ unzips DNA double helix & separates the 2 parental DNA strands
- single-stranded binding proteins bind to and stabilise separated ssDNA, keeping them apart so that they remain ss and can serve as templates for synthesis of complementary DNA strand
- Topoisomerase relieves overwinding strain ahead of replication forks by breaking, swivelling & rejoining DNA strands
Describe DNA synthesis: synthesis of new strands
- Primase catalyses synthesis of RNA primer on each parental DNA strand→ provides free 3’ OH needed for DNA polymerase to initiate DNA synthesis
- Cbp occurs between template strand and free incoming dNTPs, where A forms 2 H bonds w T and G forms 3 H bonds w C
- DNA polymerase catalyses the formation of phosphodiester bonds, linking DNA nucleotides & synthesizing the new strand in the 5’ to 3’ direction
- Anti-parallel parental strands→ 2 new strands synthesised in opposite directions
> Leading strand synthesised continuously towards replication fork
> Lagging strand synthesised discontinuously away from replication fork, forming Okazaki fragments - DNA polymerase (part of it) “proof-reads” previous region as it moves along the parental strand→ ensures proper base pairing between bases. If incorrect DNA nt added, it’s removed by DNA polymerase and replaced w correct one→ ensures fidelity of DNA seq
- Different DNA pol removes RNA primer and replaces it w DNA nucleotides
- DNA ligase catalyse formation of phosphodiester bond between nt of adjacent DNA fragments, sealing nicks in DNA
Compare DNA replication in eukaryotes and prokaryotes.
- When
- Where
- No. of Ori
- Ends at
- Rate
Describe transcription
Process: Initiation
- RNA polymerase attach to promoter of gene w aid of transcription factors (protein)→ transcription initiation complex
- RNA polymerase unzips DNA double helix (and separates the 2 strands of DNA) by breaking H bonds between complementary base pairs.
- 3’ to 5’ strand/1 strand used as template strand to synthesise complementary mRNA strand
Process: Elongation
- Free ribonucleotides will bind by cbp to nt on DNA template strand: A=U, T=A, G≡C
- RNA polymerase catalyses formation of phosphodiester bonds between free ribonucleotides→ sugar phosphate backbone
- New mRNA strand synthesised in 5’ to 3’ direction
- Region of DNA that has been transcribed reanneals, forming double helix
Process: Termination
RNA polymerase dissociate from template DNA strand after it transcribes the termination seq
ss pre-mRNA molecules released
Describe post-transcriptional modification: capping
add 5’ methylguanosine cap→ methylated guanosine nucleotide added to 5’ end of pre-mRNA as soon as ~25 nucleotides produced (protects from degradation by ribonucleases, export of mature mRNA, recognition of mRNA)
Describe post-transcriptional modification: splicing
- introns excised, exons joined
- done by spliceosome with high accuracy
Describe post-transcriptional modification: polyadenylation
Add poly A tail
- Cleaving of 3’ end of pre-mRNA by enzyme endonuclease 10-35 nt downstream of polyadenylation signal, AAUAAA (highly conserved)
- Poly-A polymerase adds many adenine nt (AMP) to 3’ end to form poly-A tail
What happens during amino acid activation?
Aminoacyl-tRNA synthetase catalyses the covalent attachment of specific aa to 3’CCA stem of a tRNA with a specific anticodon, to form aminoacyl-tRNA
Explain how correct aa is joined to tRNA
Aminoacyl-tRNA synthetases have specific AS that are complementary in 3D conformation & charge to specific aa to be attached to tRNA & specific tRNA w specific anticodon→ enzyme achieves double specificity
Describe translation
Process: Initiation
- Translation initiation factors facilitate binding of small ribosomal subunit and initiator tRNA carrying methionine to newly synthesized mRNA strand. Anticodon of initiator tRNA will cbp with start codon of mRNA.
- Binding of large ribosomal subunit→ tgt forms translation initiation complex
- This positions initiator tRNA at P site, A site vacant for incoming aminoacyl-tRNA
- GTP needed (GTP→ GDP)
Process: Elongation & Translocation
- GTP needed
- 2nd aminoacyl-tRNA w specific anticodon and corresponding aa binds to A site via cbp (GTP→ GDP)
- Peptide bond formed between adjacent aa, methionine & 2nd aa in A site, catalysed by peptidyl transferase in large subunit of ribosome: 1st aa dissociates from initiator tRNA it’s originally bound to
- Translocation (GDP→ GTP): Ribosome translocates one codon down mRNA in 5’ to 3’ direction
> 1st tRNA shifted to E site, released into cytosol and recycled
> 2nd tRNA, carrying growing polypeptide chain, shifted to P site, carries growing polypeptide
> Empty A site: receive 3rd aminoacyl-tRNA, w anticodon complementary to 3rd codon of mRNA
- Process repeats until stop/termination codon on mRNA exposed to A site on ribosome
Process: Termination of translation
- Stop codon reaches A site→ release factors enter A site→ hydrolysis of bond between polypeptide chain & tRNA in P site→ polypeptide released from ribosome & completes its folding into its necessary secondary & tertiary struc (3D conformation)
- Ribosome disassembles into its subunits
Why is simultaneous transcription and translation possible in prokaryotes?
No membrane bound nucleus (as soon as mRNA is transcribed ribosomes can attach to mRNA for translation) + mRNA no need undergo post-transcriptional modification
Compare DNA replication, transcription and translation.
- Location in cell
- Start and end at
- Things involved:
> Enzymes
> raw material
> ribosomes
> Template (S/D) - Process
> Cbp (S/D)
> Bonds
> direction - products
Name 2 mutagens
excessive UV, tar in cigarettes
Explain how gene mutations may affect the protein coded for by the gene
change in the nt sequence in DNA→ may change aa seq, which may change 3D shape and hence function of protein→ affect phenotype
Frame shift mutation–> diff aa seq–> diff and non-functional polypeptide–> diff 3D conf of protein; possibly truncated polypeptide
Substitution mutation: one nt replaced by another nt
- Change in codon–> change in aa
- same biochemical properties–> same 3D conf
- diff biochemical properties–> diff 3D conf, may not be functional
Inversion: segment of nt seq separates and rejoins at original position, but inverted
- diff codons on mRNA–> diff aa seq–> diff folding & 3D conf, possibly truncated polipeptide
What type of mutation causes sickle-cell anaemia?
Single base substitution mutation–> CTC becomes CAC
Sickle-cell anaemia: explain the significance of the change in aa to the properties of haemoglobin
- → change in 1°/aa seq in polypeptide: charged, hydrophilic glutamic acid becomes non-polar, hydrophobic valine→ change in bonds between R groups, change in 2°, 3°, 4° structure & change in conformation→ normal Hb A becomes Hb S
- → change in properties of Hb and phenotype of rbc: At low [O2], O2 released by Hb S, unusual conf change occurs→ hydrophobic patch sticks out, which attaches to another hydrophobic patch on another Hb S→ Hb S polymerises into abnormal, rigid rod-like fibres
Sickle-cell anaemia: Describe effects of the change in properties of haemoglobin
- Long insoluble fibres in rbc→ distort shape of normal biconcave rbc and make it sickle shaped
- Sickle rbc more fragile & break easily→ shorter life span→ shortage of rbc & poor oxygen transport→ anaemia, lack of energy & heart failure
- Sickle rbc pointed & elongated→ lodge in small blood vessels, interfere w blood circulation
> Deprive organs of oxygen → organ damage
> Many localised blockages→ death of surrounding tissue→ severe pain
Describe the inheritance of sickle-cell anaemia
Recessive condition, both alleles of β globin chains mutated for symptoms to appear
- homozygous recessive: 2 copies of Hb S gene → sickle cell disease
- Heterozygous individuals: Hb A & Hb S→ sickle cell trait; resistant to malaria
What kinds of structural chromosomal abberations are there?
- Deletion, duplication, inversion of a segment
- reciprocal translocation: moves a segment from one chromosome to to another, non-homologous one
When is deletion and duplication of a chromosome likely to occur? How does it occur?
During crossing over: chromatids break & rejoin at incorrect places→ one chroma give up more genes than it receives→ 1 chromo w deletion mutation and 1 w duplication mutation
→ reduced/additional genes→ phenotypic abnormalities
Define aneuploidy
cell does not have a chromosome number that is an exact multiple of haploid no.
Extra/fewer copies of chromosomes than wild types
(e.g. trisomic/monosomic)
How does non-disjuction lead to aneuploidy?
In gamete formation: homologous chromosomes don’t move properly to opp. poles at meiosis I / sister chromatids fail to separate properly to opp. poles at meiosis II
→ 1 gamete receives 2 of the same type chromosome, another gamete receives no copy
If either aberrant gametes unite w a normal gamete at fertilisation, offspring will have abnormal no. of a particular chromosome = aneuploidy. Mitosis subsequently transmits the mutation to all cells of the embryo that arose from this mutant cell
What chromosomal abberation occurs in Down syndrome/Trisomy 21?
Extra chromosome 21, each body cell w 47 chromosomes
Usually caused by non-disjunction during meiosis I
What are the physical characteristics of those with Down syndrome?
Characteristic facial features, short stature, heart defects and mental retardation. Most are sterile
