DNA Synthesis, Transcription, Translation Flashcards
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
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)
What is the structure of double stranded DNA
- Covalent phosphodiestar bonds between nucleotides
- Hydrogen bonds (two strands)
- Base pairing
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
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
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)
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)
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)
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)
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
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)
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
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
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 cognatetRNAs
- 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
What is translation
- Initiation
- mRNA and aminoacylated tRNA binds to small ribosomal subunit, large subunit binds
- Begins with met, IFs bind 40s, mediate association - 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 - Termination
- Stop codon encountered
- mRNA / protein dissociate
- Ribosomal units recycled - 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
