Past exam of the biochemistry and Molecular Biology course of the University of Cambridge / 2025 / Paper 2 / Section A

For a primary sigma factor of the ${\sigma }^{70}$ family, distinguish the following regions. Region 1.1 restrains inappropriate DNA binding by free sigma factor and must move out of the downstream-DNA channel during formation of the open RNA polymerase complex; region 1.2 contributes to recognition around the discriminator downstream of the $-10$ element. Domain 2 binds the RNA polymerase core, recognizes the $-10$ gene promoter element and helps melt the duplex and stabilize the exposed non-template strand. Domain 3 recognizes an extended $-10$ element; its region 3.2 projects toward the active-centre channel, helps position template and initiating substrates, and must be displaced as the nascent RNA grows. Domain 4 recognizes the $-35$ element through a helix-turn-helix motif and contacts the RNA polymerase beta flap and some transcription activators. Domains 2 and 4 supply the principal $-10$ and $-35$ sequence recognition, with the other regions controlling opening and productive initiation. This domain map concerns the ${\sigma }^{70}$ family, rather than every structurally distinct bacterial sigma factor.^[ref]

Incubate a labelled precursor messenger RNA containing an authentic $3^{\prime }$-processing signal with a HeLa nuclear extract, then resolve the products by denaturing polyacrylamide gel electrophoresis. With ATP, the upstream cleavage product acquires a heterogeneous poly(A) tail. Replace ATP with an appropriate non-incorporable analogue, such as alpha,beta-methylene-ATP: a discrete product ending at the normal cleavage site still appears, but the extended tail does not. Thus cleavage can occur when polyadenylation is blocked; it is not cleavage brought about by synthesis of the tail.^[ref]

Conversely, supply an RNA substrate already ending at the normal cleavage site: addition of ATP and the appropriate poly(A) polymerase machinery can extend its free $3^{\prime }$ hydroxyl without any new endonucleolytic cut.^[ref] Compare precursor, pre-cleaved substrate, no-extract and no-ATP controls, and map the product ends to distinguish correct processing from non-specific degradation. These are separable reactions, normally coordinated in one processing complex. Their separability does not imply that they use completely independent recognition factors.

A detectable burst kinetics phase requires rapid formation and detection of an initial reaction product, followed by a slower step that regenerates competent enzyme. A common hydrolysis mechanism iswhere $E^{\ast }$ can be a covalent acyl-enzyme intermediate. With excess substrate, rapid binding and $k_2\gg k_3$, the first turnover rapidly releases $P_1$ and traps much of the enzyme as $E^{\ast }$. Subsequent turnovers wait for the slow regeneration step. The measured product must report the fast step: monitoring only $P_2$ need not reveal a burst. Fast mixing and sufficient time resolution are needed, while spontaneous substrate hydrolysis, depletion and enzyme inactivation must be excluded.

For an illustrative saturated two-step cycle, initially unmodified enzyme concentration $E_T$ givesThe second term approaches a burst amplitude near $E_T$ when $k_2\gg k_3$, followed by the slower steady-state slope. A covalent intermediate is a useful example, not a logical requirement for every possible burst mechanism.

The enzyme adenylate kinase rapidly interconverts the adenine nucleotides:For approximately fixed conditions and near-equilibrium activity ratios,The AMP:ATP ratio responds quadratically to ADP:ATP. Doubling ADP:ATP therefore quadruples AMP:ATP. In working skeletal muscle, ATP is also buffered by creatine kinase and continuing ATP production, so its fractional change is relatively small. The initially smaller AMP pool can undergo a much larger fractional increase, providing a sensitive signal of energetic stress to regulators such as AMP-activated protein kinase. This concerns sensitivity of relative changes; it does not mean that the absolute AMP concentration exceeds ATP.

The mitochondrial fission-fusion cycle is continuous remodeling of a mitochondrial population by division and merger. In mammalian biological cells, mitofusins promote fusion of outer mitochondrial membranes, while OPA1 supports fusion of inner mitochondrial membranes. Fusion mixes matrix contents and permits functional complementation between mitochondria. Genetic disruption and rescue of mitofusins demonstrated their requirement for normal mitochondrial fusion.^[ref]

For fission, recruited Drp1 assembles at constrictions and uses GTP-dependent remodeling to help divide the organelle.^[ref] The balance controls network shape, distribution and quality control. Division can help distribute mitochondria to daughter biological cells and segregate damaged portions for mitophagy; fusion can restore mixed contents. These are regulated competing events in a network, not an obligatory synchronized sequence that every mitochondrion completes on a fixed timetable.

A steroid hormone crosses the lipid bilayer and binds an intracellular nuclear receptor. For a cytoplasmic glucocorticoid receptor, ligand binding alters association with molecular chaperones, exposes nuclear-targeting functions and promotes accumulation in the cell nucleus. Other steroid hormone receptors already have substantial nuclear localization. The activated receptor binds specific hormone response elements in DNA, often as a dimer, and changes recruitment of transcription coactivators or transcription corepressors. These factors alter local chromatin and recruitment or activity of the RNA polymerase II machinery. The result is selective activation or repression of target genes, followed by altered messenger RNA and protein abundance; the hormone itself does not act as an RNA polymerase or directly rewrite the DNA sequence.

Fucci uses two fluorescent protein fusions carrying cell-cycle-regulated degradation signals: a red/orange Cdt1 fragment accumulates mainly in G1 phase, whereas a green geminin fragment accumulates through S phase, G2 phase and much of M phase. Opposing activities of SCF ubiquitin ligase and anaphase-promoting complex regulate reporter degradation through the ubiquitin-proteasome system; the fluorophores report those regulated stabilities rather than simply measuring total endogenous Cdt1 and geminin.^[ref]

Live imaging follows individual biological cells through red, overlap and green states, allowing measurements of phase timing, spatial patterns and responses to perturbation without repeatedly fixing the sample. Flow cytometry can separate populations by reporter color. The basic two-color version does not independently resolve every phase, and a color transition is an indirect readout of ubiquitin ligase activity. Validate timing with DNA content or a DNA replication marker and control for altered reporter expression, fluorophore maturation and drug effects on degradation.

Two distinct possibilities are a second mutation affecting drug sensitivity of the targeted receptor tyrosine kinase, so that it remains active at the achieved inhibitor concentration, and activation of a bypass signaling route, such as another receptor tyrosine kinase or a downstream Ras or protein kinase component, restoring proliferation despite inhibition of the original target. The resistant population may arise by selection of rare pre-existing biological cells or by later acquisition of a change; recurrence alone cannot distinguish those histories. Compare target engagement, target phosphorylation and downstream signaling in the original and recurrent samples, with DNA sequencing and functional rescue or perturbation of the candidate resistance mechanism.

An antibody-drug conjugate couples an antibody against a tumor-associated surface antigen to a potent cytotoxic payload through a suitable chemical linker. Binding enriches delivery to biological cells displaying the target. Many designs undergo receptor-mediated endocytosis, trafficking through endosomes to a lysosome, and release of an active drug through linker cleavage or degradation of the antibody. The released payload can disrupt microtubules or damage DNA, killing the target biological cell. Specificity comes from targeted delivery combined with controlled payload release. It is relative rather than absolute: normal biological cells may share the antigen, and a membrane-permeable released payload can also affect neighboring biological cells. Successful action therefore depends on antigen abundance, accessibility, internalization, linker stability and payload sensitivity.

The hydrogenosomes and mitosomes are specialized mitochondrion-related organelles found in various anaerobic or oxygen-limited eukaryotes. Typical hydrogenosomes support anaerobic ATP production, converting substrates such as pyruvate into products including acetate, carbon dioxide and molecular hydrogen. Substrate-level phosphorylation supplies ATP, while hydrogenase disposes of reducing equivalents as hydrogen; oxygen is not the terminal acceptor of a conventional respiratory chain in this pathway.^[ref]

The mitosomes retain selected essential metabolic functions but do not generate ATP by oxidative phosphorylation. In Giardia, their role includes iron-sulfur cluster assembly and protein maturation, demonstrated using organelle markers and biochemical reconstitution.^[ref] Their functions are lineage-dependent: Entamoeba histolytica mitosomes compartmentalize a sulfate activation pathway.^[ref] Thus neither organelle class should be described simply as a nonfunctional mitochondrion.