DNA Synthesis, Transcription, Translation Flashcards Preview

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Flashcards in DNA Synthesis, Transcription, Translation Deck (22)
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1

What is the biological function of nucleotides and nucleic acids

- Encode DNA / RNA
- Cell signalling / transduction
- Metabolism
- Enzyme reactions
- Co-factors / co-enzymes
- Regulatory molecules

2

What is the structure of common nucleotides

- Nitrogenous base
- Phosphate (C and N atoms numbered in cyclic format)
- Pentose (C are designated N’ to alleviate confusion)

3

What is the structure of double stranded DNA

- Covalent phosphodiestar bonds between nucleotides
- Hydrogen bonds (two strands)
- Base pairing

4

What is denaturation and annealing of DNA

Denaturation:
- Covalent bonds remain intact (genetic code intact)
- H bonds broken (two strands separate)
- Base stacking is lost (UV absorbance increases), not uniform
- CG requires more energy (3H) to break than AT (2H)
- Fundamental principle of PCR (ability to separate H bonds and re-anneal due to complementarity)
Annealing
- Reversibility of denaturation
- Recombine DNA in double stranded form

5

What is the mechanism of DNA replication

- “Parental” double-stranded DNA molecule is converted to two identical molecules, one strand serves as a template for the production of a second strand
- Semiconservative, bi-directionally, synthesis occurs in 5’ → 3’ direction (read in 3’ → 5’), semi-discontinuous
- Leading Strand: Continuously synthesised as the replication fork advances
- Lagging Strand: Discontinuously synthesised in short pieces (okazaki fragments) that are later joined

6

What are the principles of DNA damage and repair

- Chemical reactions and physical processes damage DNA, majority are corrected using undamaged strand
- Mutations occur when these damages escape repair
- Lesions (mutation), mismatches, abnormal bases (spontaneous deamination), pyrimidine dimers (UV) and backbone lesions (radiation)

7

What are the structures of key RNAs (mRNA, tRNA, rRNA)

- mRNA: Encode AA sequences of all polypeptides found in cell, transcription is complex, relies on protein-protein contacts, highly conserved transcription factors
- tRNA: Match anticodon to mRNA while carrying a specific AA used for protein synthesis
- rRNA: Constituents of large and small ribosomal subunits
- microRNA: Regulate the expression of genes, possibly via binding to specific nucleotide sequences
- Ribozymes: Catalytic RNA molecules that act as enzymes, often use metal ion cofactors (group I introns)

8

What is transcription and the steps involved

- DNA dependent synthesis of RNA, tightly regulated to control concentration of proteins
1. RNA polymerase binds promoter and unwinds DNA (forms bubble of ~17 bp)
2. RNA polymerase binds triphosphate nucleosides and generates RNA transcript via complementary base pairing
3. Site of synthesis moves along DNA away from promotor generating +ve supercoils ahead and -ve supercoils behind (relaxed by topoisomerase)
4. DNA that has been transcribed recoils
5. Transcription reaches terminator, p-independent (hairpin) or p-dependent (protein)

9

What is capping, polyA tail and splicing (processing of mRNA)

- Primary RNA transcript in eukaryotes requires processing before it becomes messenger RNA
- 5’ Cap: 7-methylguanosine links to 5’-end, formed with a molecule of GTP, protects RNA from nucleases, forms a binding site for ribosome
- Poly(A) Tail: Binding site on mRNA, protects mRNA from degradation, RNA Pol II synthesises RNA beyond cleavage signal sequence
- Splicing: Introns (non coding) are removed for mature RNA (50-700,000 bp), exons (coding for genes) are kept (<1000 bp)

10

What is the difference between the template and coding strand in transcription

- Template: Serves as template for RNA polymerase (strand being copied into RNA)
- Coding: Non-template strand, same sequence as the RNA transcript (codes for protein), regulatory sequences contained on this strand

11

What are the principles and purpose of promoter regions

- Determine the transcription start site and direct binding of RNA poly (TATA box in rapidly transcribed genes)
- Located near the start site, general TF and RNA pol assemble (similar for all genes)

12

What is genetic code

- Set of rules that determines how a nucleotide sequence is converted into amino acid sequence of a protein
- Complementary structure allows precise replication during cell division
- First codon establishes reading frame, written in 5’ to 3’ direction
- Start / Stop: Translation begins at start codon (AUG) and ends at stop codon (UAA, UAG, UGA)
- Non-Overlapping: Codons do not share nucleotides, increased flexibility
- Redundancy: Most AA have more than one codon, some are less subject to mutation because of degeneracy / abundance of tRNAs, 20 amino acids with 61 possible codons

13

What are the 4 main types of mutations

Silent Mutation:
- Change in DNA base sequence causes no change in the activity of the product encoded by the gene, because of degeneracy / redundancy new codon might still code for the same AA
- Nucleotide is substituted, often corresponding to third position of mRNA codon
- Synonymous, protein function unaffected
Missense Mutation:
- Incorrect base may cause the insertion of an incorrect amino acid in the protein
- Single nucleotide base is replaced with a different base, resulting in AA substitution
- Non-Synonymous, can result in non-functional protein
Nonsense Mutation:
- Prevents synthesis of a complete functional protein, only a fragment is synthesised, result in a truncated and non functional protein
- Nonsense codon, premature termination and non functional protein
Frameshift Mutation:
- One or more nucleotide pairs are deleted or inserted in the DNA, shifts translational reading frame
- Leads to different consecutive amino acids, several are changed / incorrect, premature termination

14

Describe the synthesis of aminoacyl-tRNA (activation)

- Activation of AA, creation of aminoacyl intermediate (tRNA is aminoacylated)
- Aminoacyl-tRNA synthetases are enzymes that catalyse covalent attachment of AA to cognate tRNAs
- Aminoacyl-tRNA synthetases esterify 20 AA to corresponding tRNAs, requires energy
- ATP creates aminoacyladenylate intermediate, pyrophosphate (PPi) is cleaved, (ATP → AMP)
- Each specific AA is bound to the matching tRNA
- Most cells contain 20 different aminoacyl-tRNA synthetases, one for each AA

15

What is translation

1. Initiation
- mRNA and aminoacylated tRNA binds to small ribosomal subunit, large subunit binds
- Begins with met, IFs bind 40s, mediate association
2. Elongation
- Binding of aminoacyl tRNA to elongation factor (GTP complex)
- Successive cycles occur to form AA
- Peptide bonds form, catalysed by ribozymes
- Movement from A to P to E sites
3. Termination
- Stop codon encountered
- mRNA / protein dissociate
- Ribosomal units recycled
4. Post-Translational Modifications
- Enzymatic removal of formyl group, met or residues
- Acetylation of N terminal residue
- Removing sequence to activate enzyme
- Glycoprotein linking
- Sorting of proteins, targeted for and imported / exported

16

What is protein targeting, signal sequencing and degradation

- Biological mechanism by which proteins are transported to appropriate destinations in the cell or outside it
- Signal Sequence must be near N-terminus, direct insertion of proteins into membrane of ER
- Two major pathways for degradation (ubiquitin-proteasome pathway or lysosomal proteolysis)

17

What are the 7 methods of regulating protein concentration in the cell

- Control of synthesis of primary RNA transcript (73%)
- Post-transcriptional modifications of mRNA
- Degradation of mRNA (11%)
- Protein synthesis and translational control (8%)
- Post-translational modification of protein
- Targeting and transport of the protein
- Degradation of protein (8%)

18

What are DNA elements that control transcription

- Cis-Acting Elements: DNA sequences required in vicinity of gene to be expressed
- Promoters: determine transcription start site
- Regulatory Sequences: Binding sites for proteins
- Enhancers / Repressors: Regulate particular gene
- Transcription Factors: Bind to regulatory elements of gene, constitutive (house keeping genes, all cells) or regulated (certain tissues only)

19

What are protein factors that control transcription

- Enhancers / Repressors: Help regulate a particular gene, often tissue specific and function only in specific differentiated cell types, may be many kb to 100s of kb away from the transcriptional start site
- Repressor: Reduce RNA Pol-promoter interactions or block the polymerase, bind to operator sequences on DNA, usually near a promoter in bacteria but further away in many eukaryotes
- Effector: Can bind to repressor and induce a conformational change, change may increase or decrease repressor’s affinity for the operator and thus may increase or decrease transcription

20

How is protein synthesis regulated (splicing)

- Constitutive: Consecutive exons are kept
- Alternative: Only some exons are kept, troponin
- Transport / localisation affects ability to function in particular areas

21

What are the stages of DNA replication

1. Topoisomerase / gyrase relaxes supercoiling and unwinds DNA
2. DNA polymerase synthesises leading strand continuously from the primer in 5' 3' direction towards replication fork
3. Lagging strand synthesised discontinuously in 5' 3' away from replication fork
4. Primase (RNA primer) is digested by DNA polymerase and replaces it with nucleotides
5. DNA ligase joins discontinuous okazaki fragments of lagging strand

22

What are methods of translational control

- Phosphorylation of translation initiation factors, translational repressors, mediated regulation (silencing)
- Ferritin: Fe binding storage protein
- Low Fe: Don’t store Fe, stops ferritin synthesis, iron responsive binding protein (IRB) binds iron response element (IRE) and blocks ribosomes
- High Fe: Need to store Fe increases ferritin, control is at level of translation, IRB doesn’t bind IRE, ribosomes translates mRNA