Molecular Biology Mock Exam Flashcards

Early Discoveries in Genetics and Molecular Biology

• The Discovery of Bacterial Transformation:     * The process of bacterial transformation (the ability of a bacteria to alter its genetic makeup by taking up foreign $DNA$ from its surroundings) was originally discovered by Griffith.     * This discovery laid the groundwork for identifying the "transforming principle."

• The Hershey and Chase Experiment:     * Utilized $T2$ bacteriophages and radioactive labeling ($^{32}P$ to label $DNA$ and $^{35}S$ to label proteins).     * Conclusion: The experiment demonstrated that $DNA$, not protein, functions as the genetic material.     * It effectively refuted the idea that the phage coat (protein) contains the genetic material required for viral replication.

• Other Key Figures:     * Chargaff: Discovered the rules of base pairing ($A=T$ and $G=C$).     * Meselson and Stahl: Proved that $DNA$ replication is semiconservative using nitrogen isotope labeling.

Principles of Mendelian Inheritance

• Allele Representation:     * Dominant alleles are signified by upper case letters.     * Recessive alleles are signified by lower case letters.

• Trait Selection (Pisum sativum model):     * Flower Color: Purple ($P$) is dominant; White ($p$) is recessive.     * Plant Height: Tall ($T$) is dominant; Dwarf ($t$) is recessive.

• Genotype to Phenotype Analysis:     * A plant with the genotype $PpTt$ is a double heterozygote.     * Because it possesses at least one dominant allele for each trait ($P$ and $T$), the expressed phenotype is purple flowers, tall.

Molecular Mechanisms of DNA Replication

• Okazaki Fragments:     * These are segments of $DNA$ that act as intermediates in the synthesis of the lagging strand.     * Synthesis of the lagging strand is discontinuous because $DNA$ polymerase can only synthesize in the $5' \rightarrow 3'$ direction, forcing it to work away from the replication fork in short bursts.

• Initiation Requirements in $E. coli$:     * Essential components for initiation include:         * $DnaB$ (helicase): Unwinds the double helix at the origin.         * $DnaG$ (primase): Synthesizes short $RNA$ primers required for $DNA$ polymerase to start.         * Dam methylase: Methylates the $DNA$ at specific sequences ($GATC$ sites) to regulate replication timing and coordination.     * Note: $DNA$ ligase is not required for the initiation of replication; it is required later to seal nicks between fragments.

• $E. coli$ DNA Polymerase I:     * Functions primarily in clean-up and repair during replication.     * Possesses a unique $5' \rightarrow 3'$ exonuclease activity.     * This activity is specifically involved in the removal of $RNA$ primers by nick translation, allowing the enzyme to simultaneously remove the primer and fill the gap with $DNA$.

• Prokaryotic DNA Polymerase III:     * The primary replicative enzyme in $E. coli$.     * Features a $\beta$ subunit (beta clamp), which acts as a circular clamp that encircles the $DNA$.     * Function: Dramatically improves the processivity of $DNA$ synthesis (the number of nucleotides added before the polymerase dissociates from the template).

Mechanisms and Regulation of Transcription in Prokaryotes

• E. coli RNA Polymerase Structure and Function:     * The enzyme functions as a holoenzyme, which consists of several subunits including the core enzyme and the sigma ($\sigma$) factor.     * Core Enzyme (\alpha_2 ̀̂ \beta \beta' ̀́ \omega): It is capable of polymerizing $RNA$ but lacks specific binding to promoter regions.     * Sigma Factor ($\sigma$): Required for the selective binding to promoter regions and the initiation of synthesis; it falls off after initiation.     * Critical attributes: The enzyme uses nucleoside $5'-triphosphates$ (ATP, ̀̂ GTP, ̀́ CTP, ̀́ UTP) as substrates and produces an $RNA$ product that hybridizes with the $DNA$ template strand.

• Transcription Initiation Sequence:     * The correct chronological order of events is:         1. Closed complex formation: Initial binding of holoenzyme to the promoter ($DNA$ remains double-stranded).         2. Open complex formation: Melting of the $DNA$ helix to create a transcription bubble.         3. Start of $RNA$ synthesis: Polymerization of the first few nucleotides.         4. Promoter clearance: The polymerase breaks its hold on the promoter and moves down the template, often releasing the sigma factor.

• Transcription Termination (Rho-dependent):     * \rho ̀̂ ́ \text{-terminator}: A protein factor with $ATP$-dependent $RNA-DNA$ helicase activity.     * Directionality: It migrates in the $5' \rightarrow 3'$ direction along the nascent $RNA$ (Option B in the exam stating it moves $3' \rightarrow 5'$ is false).     * Mechanism: It causes the release of $RNA$ polymerase when it reaches a $CA$ -rich sequence near the end of the transcript, utilizing $ATP$ hydrolysis to power its movement and displacement.

Protein Synthesis and The Translation Apparatus

• mRNA Coding Capacity and Polypeptide Weight:     * Given: A bacterial $mRNA$ of $800 \text{ nucleotides}$.     * Assumption: Average amino acid residue molecular weight is $110$.     * Calculation:         * Maximum possible codons: $800 / 3 \approx 266 \text{ codons}$.         * Molecular weight: $266 \text{ amino acids} \times 110 \text{ weight/amino acid} = 29,260$.         * The largest polypeptide would have a weight of approximately $30,000$.

• The Wobble Hypothesis:     * Proposed by Francis Crick to explain why there are fewer than 61 $tRNAs$ for 61 codons.     * Key feature: The "wobble" or non-standard base pairing occurs only in the first base of the anticodon (corresponding to the third base of the codon).

• tRNA Structure and Characteristics:     * Contains more than just the standard A, C, G ̀̂, ̀́ U bases; it includes many modified bases (e.g., pseudouridine, inosine).     * Single-stranded but folds into a secondary structure with short, double-helical regions (cloverleaf model).     * The amino acid is always attached to an $A$ nucleotide at the $3'$ end ($CCA$ tail).     * There is at least one specific $tRNA$ for each of the 20 standard amino acids.

• Ribosomal Initiation in Bacteria:     * Required: $mRNA$, $formylmethionyl tRNA_{f}^{Met}$ (fMet ̀̂ ́ ̀́ ́ ́ ́ ́ ́ ́ - tRNA_{f}^{Met}), and Initiation Factor 2 ($IF-2$).     * Not Required: $EF-Tu$, which is an elongation factor used for bringing aminoacyl-tRNAs to the ribosome after initiation is complete.

• Protein Elongation Phase:     * During elongation, peptidyl transferase acts as the catalyst for peptide bond formation.     * Peptidyl transferase is a ribozyme (the catalytic activity resides in the high-molecular-weight $rRNA$ of the large ribosomal subunit, not a protein).     * Note: Incoming aminoacylated $tRNAs$ bind to the A site, not the P site.

Bacterial Gene Regulation: Operons and Attenuation

• Housekeeping (Constitutive) Genes:     * These genes are expressed constantly.     * Variation in their expression levels is primarily due to different promoter affinities for the $RNA$ polymerase holoenzyme. Stronger promoters bind the enzyme more frequently.

• Promoter Characteristics in $E. coli$:     * Many promoters share structural similarities and resemble a consensus sequence.     * The closer a sequence is to the consensus, the higher its affinity for $RNA$ polymerase holoenzyme.

• The Tryptophan ($trp$) Operon:     * Attenuation: A regulatory mechanism involving the leader peptide sequence. Mutating selected bases in sequence 3 of the leader peptide prevents the formation of the required hairpin structures, resulting in decreased attenuation.     * Repression: The $trp$ operon is repressed when tryptophan is abundant. The tryptophan repressor binds to the $trp$ operator only in the presence of tryptophan (tryptophan serves as a co-repressor).

• The Lac Operon and CRP:     * $CRP$ (cAMP receptor protein), also known as $CAP$ (Catabolite Activator Protein), binds to $DNA$ near the lac promoter.     * Function: It assists $RNA$ polymerase binding to the lac promoter to increase transcription.     * Regulation: Binding occurs only when $cAMP$ levels are high (which occurs when glucose levels are low).

DNA Repair Mechanisms and Mutagenicity Testing

• Methyl-Directed Mismatch Repair ($MMR$):     * Involves identifying and repairing mismatched bases in newly synthesized $DNA$.     * Key enzymes: $DNA$ helicase II, $DNA$ ligase, and $DNA$ polymerase III.     * $DNA$ glycosylase is not involved in $MMR$; it is a primary component of Base-Excision Repair ($BER$).

• Nucleotide-Excision Repair ($NER$):     * The $ABC$ excinuclease is essential for this pathway, which removes bulky lesions like those caused by UV radiation.

• The Ames Test:     * A biological assay used specifically to measure the mutagenic effects of various chemical compounds. It uses a strain of Salmonella typhimurium to see if a chemical can cause a reversion of a mutation.

• Thymidine Dimer Repair:     * Thymidine dimers (caused by UV light) can be repaired via direct repair (e.g., photoreactivation using the enzyme DNA photolyase) or by nucleotide-excision repair.

Comprehensive Mock MCQ Exam: Question and Answer Compendium

• Questions and Discussion (Key Findings from Examination):     * 1) Who discovered bacterial transformation? -> Griffith.     * 2) Hershey and Chase conclusion? -> $DNA$ is the genetic material.     * 3) Phenotype of $PpTt$? -> Purple, tall.     * 4) What is an Okazaki fragment? -> Lagging strand synthesis intermediate.     * 5) Not required for $E. coli$ replication initiation? -> $DNA$ ligase.     * 6) $5' - 3'$ exonuclease of Pol I? -> Primer removal by nick translation.     * 7) Prokaryotic Pol III clamp? -> $\beta$ subunit improves processivity.     * 8) False statement about $RNA$ polymerase? -> Core enzyme binds promoter selectively (False; sigma is needed).     * 9) Order of initiation? -> Closed, Open, Synthesis, Clearance.     * 10) False about core enzyme? -> No catalytic activity unless sigma bound (False; it has activity, just lacks specificity).     * 11) False about rho-terminator? -> Migrates $3' - 5'$ (False; it moves $5' - 3'$).     * 12) Polypeptide weight from $800$ nucleotides? -> $30,000$.     * 13) Feature of wobble hypothesis? -> Wobble occurs in first base of anticodon.     * 14) False about $tRNA$? -> $A, C, G, U$ are the only bases (False).     * 15) Not required for ribosomal initiation? -> $EF-Tu$.     * 16) True about elongation? -> Peptidyl transferase is a ribozyme.     * 17) Mechanism for constitutive enzyme variation? -> Different promoter affinities.     * 18) $E. coli$ promoter description? -> Many resemble a consensus sequence.     * 19) Mutating sequence 3 in $trp$ leader? -> Attenuation is decreased.     * 20) $trp$ repressor behavior? -> Binds operator in presence of tryptophan.     * 21) $CRP$ binding in lac operon? -> Assists $RNA$ polymerase binding.     * 22) Not involved in mismatch repair? -> $DNA$ glycosylase.     * 23) $ABC$ excinuclease role? -> Nucleotide-excision repair.     * 24) Ames test purpose? -> Measure mutagenic effect of chemicals.     * 25) Repair for thymidine dimers? -> Direct repair.