🧬 DNA & RNA Visualization

✅ Answer:

Gene: A gene is a specific segment of DNA that contains the instructions for making a particular protein or functional RNA molecule. It is the basic unit of heredity.

Genome: The genome is the complete set of genetic material (DNA) in an organism, including all genes and non-coding sequences. It contains all the information needed to build and maintain that organism.

✅ Answer:

Promoter: A DNA sequence where RNA polymerase binds to initiate transcription. It determines which strand is transcribed and the direction of transcription. Contains -10 (Pribnow box) and -35 consensus sequences in prokaryotes.

Operator: A regulatory DNA sequence located between the promoter and structural genes. It acts as a binding site for repressor proteins. When repressor binds, it blocks RNA polymerase, preventing transcription.

✅ Answer:

Aminoacyl-tRNA synthetase is the enzyme responsible for charging tRNA. It catalyzes the attachment of a specific amino acid to its corresponding tRNA molecule in a two-step reaction:

• Amino acid + ATP → Aminoacyl-AMP + PPi
• Aminoacyl-AMP + tRNA → Aminoacyl-tRNA + AMP

There are 20 different aminoacyl-tRNA synthetases, one for each amino acid.

✅ Answer:

Open Reading Frame (ORF) is a sequence of DNA or RNA that begins with a start codon (AUG) and ends with a stop codon (UAA, UAG, or UGA), with no intervening stop codons. It represents a region that potentially encodes a protein.

The reading frame is determined by the position of the first nucleotide, and DNA can be read in three different frames on each strand (6 total reading frames).

✅ Answer:

Streptomycin - It binds to the 30S ribosomal subunit and causes misreading of mRNA, leading to incorporation of incorrect amino acids into the polypeptide chain.

Other examples: Tetracycline (blocks A-site), Chloramphenicol (inhibits peptidyl transferase in 50S), Erythromycin (blocks exit tunnel in 50S).

✅ Answer:

Genetic code is the set of rules by which information encoded in genetic material (DNA or RNA) is translated into proteins by living cells. It defines how sequences of three nucleotides (codons) correspond to specific amino acids.

Properties: Triplet (3 bases = 1 codon), Degenerate (multiple codons for same amino acid), Non-overlapping, Universal (same in most organisms), Unambiguous (each codon specifies only one amino acid).

✅ Answer:

Consensus sequence is a sequence of nucleotides that shows the most common bases found at each position when comparing related sequences from different genes or organisms. It represents the "average" or most typical sequence.

Example: The Pribnow box in prokaryotic promoters has the consensus sequence TATAAT (located at -10 position). The -35 region has consensus sequence TTGACA.

✅ Answer:

Alternative splicing is a process by which a single pre-mRNA can be spliced in different ways to produce multiple different mature mRNA variants, which can then be translated into different protein isoforms.

This allows one gene to encode multiple proteins, increasing proteome diversity without increasing genome size. Types include: exon skipping, intron retention, alternative 5' or 3' splice sites, and mutually exclusive exons.

✅ Answer:

Salient Features of Watson-Crick DNA Model:

• DNA is a double helix consisting of two antiparallel polynucleotide strands
• The two strands wind around a common axis in a right-handed manner
• The sugar-phosphate backbone is on the outside, bases are inside
• Bases are paired by hydrogen bonds: A-T (2 H-bonds), G-C (3 H-bonds)
• The helix has a diameter of 20Å (2 nm)
• One complete turn is 34Å with 10 base pairs per turn (3.4Å per bp)
• Has major and minor grooves

B-DNA vs Z-DNA:

• B-DNA: Right-handed helix, 10 bp/turn, most common form under physiological conditions
• Z-DNA: Left-handed helix, 12 bp/turn, zigzag backbone pattern, found in alternating purine-pyrimidine sequences

✅ Answer:

Prokaryotic Transcription:

• Initiation: RNA polymerase holoenzyme (core + σ factor) binds to promoter at -10 (TATAAT) and -35 (TTGACA) regions. DNA unwinds to form open complex.
• Elongation: σ factor releases. Core enzyme synthesizes RNA 5'→3' using template strand 3'→5'. Transcription bubble moves along DNA.
• Termination: Two mechanisms - Rho-independent (intrinsic) uses hairpin loop + poly-U stretch; Rho-dependent uses Rho protein helicase.

Mitochondrial DNA (mtDNA):

• Circular, double-stranded DNA (~16.5 kb in humans)
• Encodes 13 proteins, 22 tRNAs, and 2 rRNAs
• Maternally inherited (no recombination)
• Has its own genetic code (slightly different from nuclear code)
• High mutation rate due to lack of histone protection

✅ Answer:

Rolling Circle Replication:

• Used by some viruses (φX174), plasmids, and during bacterial conjugation
• A nick is made in one strand of circular dsDNA
• The 3'-OH end serves as primer; DNA polymerase extends it
• As new strand is synthesized, the old strand is displaced
• The displaced strand can be used as template for complementary synthesis
• Can generate multiple copies (concatamers) from one circular template

Role of DNA Methylation in Replication:

• Helps distinguish between parental (methylated) and newly synthesized (unmethylated) strands
• Important in mismatch repair - repair machinery removes bases from unmethylated strand
• Dam methylase adds methyl groups to adenine in GATC sequences

✅ Answer:

i. Base Excision Repair (BER):

• Repairs damaged or modified single bases (oxidation, alkylation, deamination)
• DNA glycosylase recognizes and removes damaged base, creating AP site
• AP endonuclease cleaves phosphodiester backbone
• DNA polymerase fills gap; ligase seals nick

ii. Topoisomerase:

• Enzymes that control DNA topology by changing supercoiling
• Type I: Cuts one strand, relaxes supercoils, religates (ATP-independent)
• Type II (DNA gyrase): Cuts both strands, passes another segment through, introduces negative supercoils (ATP-dependent)

iii. Cot Curve:

• Graph showing reassociation kinetics of denatured DNA
• Cot = initial concentration × time
• Reveals genome complexity: repetitive DNA reassociates faster than unique sequences
• Cot½ value indicates complexity - higher Cot½ means more complex genome

✅ Answer:

Initiation of Translation in Prokaryotes:

• Step 1: 30S subunit binds IF1 and IF3 (prevents premature 50S joining)
• Step 2: mRNA binds via Shine-Dalgarno sequence (AGGAGG) complementary to 16S rRNA
• Step 3: Start codon AUG positioned at P-site
• Step 4: IF2-GTP brings fMet-tRNAfMet to P-site
• Step 5: 50S subunit joins, GTP hydrolysis, IF1, IF2, IF3 release
• Result: 70S initiation complex with fMet-tRNA in P-site, A-site ready

RNA Splicing:

• Removal of introns from pre-mRNA to form mature mRNA
• Splice sites: 5' (GU) donor and 3' (AG) acceptor sites
• Branch point adenine forms lariat structure
• Mechanism: Two transesterification reactions catalyzed by spliceosome
• Spliceosome: Complex of snRNPs (U1, U2, U4, U5, U6) and proteins
• U1 binds 5' splice site, U2 binds branch point, catalysis occurs via U2-U6 pairing

✅ Answer:

Semi-conservative Replication:

• Each daughter DNA molecule contains one parental strand and one newly synthesized strand
• Proved by Meselson-Stahl experiment (1958) using ¹⁵N and ¹⁴N density labeling
• After one generation: hybrid (intermediate density); After two: 50% hybrid + 50% light

Enzymes and Proteins in DNA Replication:

• Helicase (DnaB): Unwinds double helix at replication fork
• SSB proteins: Stabilize single-stranded DNA, prevent reannealing
• Topoisomerase/Gyrase: Relieves torsional strain ahead of fork
• Primase (DnaG): Synthesizes RNA primers
• DNA Polymerase III: Main replicative enzyme, 5'→3' synthesis, proofreading
• DNA Polymerase I: Removes primers, fills gaps, exonuclease activity
• DNA Ligase: Joins Okazaki fragments by forming phosphodiester bonds
• Sliding clamp (β-clamp): Increases processivity of Pol III

Role of siRNA:

• Small interfering RNA (~21-23 nt) involved in RNA interference (RNAi)
• Guides RISC complex to complementary mRNA for degradation
• Used for gene silencing/knockdown in research and potential therapeutics