University of Calcutta

B.A./B.Sc. SEM VI Honours Examination 2024 (CBCS)

CEMA DSE-B4

Biological Polymerization:
Advantage of being 'out-of-equilibrium'

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Introduction:
The central dogma of molecular biology is a framework that describes the flow of genetic
information within a biological system. This concept was first articulated by Francis Crick in
1958 [1,2], and it highlights the directional pathway through which genetic information is
transferred from DNA to RNA to protein. It emphasizes the roles of these molecules in gene
expression and the functional activities of cells.
Remarkably, all the biological polymerization processes contained in central dogma are highly
accurate [3-7]. The following table shows some characteristic values establishing this feature.

Process Error
Translation ~10-4 - 10-3
Transcription ~10-5 - 10-4
DNA replication ~10-10- 10-8

The high accuracy is crucial as failure to achieve error-free polymerization affects the cell
survival and desired development with proper structure-function relationships. For example,
mutations affecting the accuracy of translation increase the number of unfolded proteins
leading to apoptosis or cell death [8] and to erroneous replication of genetic material [9].

In each stage of the information flow, there are various intricate mechanisms of error correction.
Still, the substrates are too similar to account for such low errors based on thermodynamic
equilibrium binding free energy differences [10]. This was a puzzle until a general mechanism
that explains such low error values called Kinetic Proofreading (KPR) was introduced by
independently by John Hopfield [11] and Jacques Ninio [12] (actually Ninio denoted the
mechanism as kinetic amplification). KPR is a non-equilibrium mechanism that effectively
multiply the small discriminations in the different steps of polymerizations to large values that
ultimately leads to high accuracy. Interestingly, such enhancement of accuracy is not possible
with the processes occurring in thermodynamic equilibrium, hence the term ‘kinetic’.
However, to operate out of equilibrium, such processes require constant energy
consumption [11]. To achieve that, they consume energy-rich molecules, like ATP, the energy
currency of the cell [13,14], to provide for the necessary driving to sustain the system in a non-
equilibrium steady state. This theoretically proposed mechanism of KPR was later
experimentally verified in various biological systems [15-19].

1. DNA Replication:
Overview: DNA replication is the process by which a double-stranded DNA molecule is copied
to produce two identical DNA molecules. This is crucial for cell division and the inheritance
of genetic material.

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Mechanism: The replication process begins at specific locations in the DNA, known as origins
of replication. Enzymes such as helicases unwind the DNA helix, and DNA polymerases
synthesize new strands by adding nucleotides complementary to the original strands.
Proofreading and Error Correction: DNA polymerases have proofreading abilities to ensure
high fidelity during replication, correcting any mismatched bases to prevent mutations. E
denotes the T7 DNAP enzyme in complex with a DNA primer template. The R and W substrates
are correct and incorrect base-paired dNTP molecules, respectively. Step 1 generates enzyme–
DNA complexes ER (or EW) with the primer elongated by one nucleotide. Addition of another
correct nucleotide to ER(EW) gives rise to PR (PW). ER* and EW* complexes denote the
primer shifted to the exonuclease site (Exo) from the polymerase site (Pol) of DNAP. This
commences proofreading in step 2. Excision of the nucleotide in step 3 resets the system to its
initial state.

2. Transcription:
Overview: During transcription, the DNA sequence of a gene is transcribed to produce a
complementary RNA strand. This RNA strand is called messenger RNA (mRNA), which
carries the genetic code from the DNA in the nucleus to the ribosomes in the cytoplasm.
Process: Transcription involves three main stages: initiation, elongation, and termination. RNA
polymerase binds to the promoter region of the gene, synthesizes the RNA strand by adding
ribonucleotides, and eventually releases the completed mRNA strand when it reaches a
termination signal.
Post-Transcriptional Modifications: In eukaryotes, the pre-mRNA undergoes splicing to
remove introns, addition of a 5’ cap, and a poly-A tail to produce mature mRNA ready for
translation.

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3. Translation:
Overview: Translation is the process by which the mRNA sequence is decoded to synthesize a
specific protein. Ribosomes facilitate this process, ensuring that the sequence of amino acids
in the polypeptide chain matches the sequence of codons in the mRNA.
Mechanism: The ribosome reads the mRNA codons in sets of three nucleotides. Transfer RNA
(tRNA) molecules, each carrying a specific amino acid, recognize and bind to the codons
through their anticodon regions. The ribosome catalyses the formation of peptide bonds
between the amino acids, elongating the polypeptide chain. Cognate (near-cognate) aa-tRNAs
in ternary complex with elongation factor Tu (EFTu) and GTP bind with ribosome in step 1 to
form ER (EW). GTP hydrolysis in step 2 results in the complex ER* (EW*). The latter can
take one of two routes. It can progress to the product PR (P W) with the elongation of the
peptide chain by one amino acid. Alternatively, it can dissociate in the proofreading step (step
3), rejecting the aa-tRNA.

Post-Translational Modifications: Newly synthesized proteins often undergo modifications

such as folding, phosphorylation, and glycosylation to become fully functional.
Conclusion:
The central dogma illustrates the flow of genetic information and emphasizes the roles of DNA,
RNA, and proteins in gene expression. Understanding this process is fundamental to molecular
biology and genetics, providing insights into cellular functions, genetic inheritance, and the
molecular basis of diseases.
Biological Error Correction Mechanisms
Introduction:
Error correction mechanisms are vital for maintaining the integrity of genetic information
during processes such as DNA replication, transcription, and translation. These mechanisms
ensure the fidelity of genetic information transfer and minimize mutations, which can lead to
diseases and malfunctions.

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1. DNA Polymerase Proofreading:
Mechanism: DNA polymerases have an intrinsic proofreading function that detects and corrects
errors during DNA replication. When an incorrect nucleotide is incorporated, the polymerase
stalls, allowing its 3' to 5' exonuclease activity to remove the mismatched nucleotide.
Importance: This proofreading ability significantly reduces the error rate during DNA
replication, ensuring the accurate duplication of the genome.
2. Mismatch Repair:
Mechanism: Mismatch repair (MMR) systems detect and repair mismatched nucleotides that
escape proofreading during DNA replication. Proteins such as MutS, MutL, and MutH in
prokaryotes (and their homologs in eukaryotes) recognize and excise the mismatched segment,
allowing DNA polymerase to fill in the correct nucleotides.
Impact on Health: Defects in mismatch repair genes are associated with an increased risk of
certain cancers, particularly colorectal cancer, highlighting the critical role of MMR in
maintaining genomic stability.
3. RNA Splicing and Editing:
Splicing: During RNA splicing, non-coding introns are removed from the pre-mRNA, and
exons are joined together to form mature mRNA. This process is facilitated by the spliceosome
complex.
RNA Editing: RNA editing involves the alteration of nucleotide sequences within an RNA
molecule, often through deamination processes that convert adenine to inosine or cytosine to
uracil. These modifications can change the coding potential of the mRNA.
Functional Significance: Accurate splicing and editing are crucial for producing functional
proteins and for the regulation of gene expression.
4. Translation Accuracy:
Mechanism: Ribosomes, tRNAs, and associated factors work together to ensure accurate
translation. Aminoacyl-tRNA synthetases play a key role by charging tRNAs with the correct
amino acids, while ribosomes use kinetic proofreading to select the correct tRNA during codon-
anticodon pairing.
Quality Control: Mechanisms such as the surveillance of mRNA quality (e.g., nonsense-
mediated decay) help prevent the translation of defective mRNAs, thereby reducing errors in
protein synthesis.
Conclusion:
Biological error correction mechanisms are essential for preserving the accuracy of genetic
information. These mechanisms help prevent mutations and ensure proper cellular function and
organismal health. Understanding these processes provides insights into the prevention and
treatment of genetic diseases.

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Cycle with Two Intermediates
Introduction:
Metabolic cycles are series of biochemical reactions that produce intermediates and regenerate
starting materials. A cycle with two intermediates is a fundamental concept in metabolic
pathways, exemplified by various biochemical processes. These cycles play a crucial role in
energy production and the synthesis of key biomolecules.

1. Example of a Metabolic
Cycle:
Glycolysis: One example of a
metabolic cycle with two
intermediates is the conversion
of glucose to pyruvate in
glycolysis, involving the
intermediates glyceraldehyde-3-
phosphate (G3P) and
phosphoenolpyruvate (PEP).
Overview of Steps: Glycolysis
consists of ten enzyme-
catalyzed steps, where glucose
is broken down into two molecules of pyruvate, producing ATP and NADH in the process.
2. Role of Intermediates:
Intermediates I1 and I2: In the glycolysis pathway, G3P (I1) and PEP (I2) are crucial
intermediates. G3P is produced in the earlier steps of glycolysis and is further metabolized to
produce PEP, which is then converted to pyruvate in the final steps.
Function: These intermediates facilitate the stepwise breakdown of glucose, allowing for the
controlled release of energy and the production of ATP.
3. Regulation of the Cycle:
Feedback Inhibition: Glycolysis is tightly regulated by feedback inhibition, where the
accumulation of ATP and other end products inhibits the activity of key enzymes such as
phosphofructokinase-1 (PFK-1).
Allosteric Regulation: Enzymes in the glycolytic pathway are regulated allosterically by
various metabolites, ensuring that the pathway is responsive to the energy needs of the cell.
Conclusion:
Metabolic cycles with two intermediates are crucial for cellular function, enabling efficient
conversion of molecules and maintaining metabolic homeostasis. Understanding these cycles
provides insights into energy production, cellular respiration, and the synthesis of vital
biomolecules.

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Cycle in Equilibrium
Introduction:
Biochemical cycles often reach a state of dynamic equilibrium where the concentrations of
reactants and products remain constant over time. Understanding cycles in equilibrium is
essential for grasping how metabolic pathways are regulated and how cells maintain
homeostasis.
1. Dynamic Equilibrium:
Definition: In a biochemical cycle, dynamic equilibrium occurs when the rate of formation of
intermediates and products equals the rate of their consumption. This balance maintains steady-
state conditions within the cell.
Importance: Dynamic equilibrium allows cells to efficiently manage resources and respond to
changes in metabolic demands without depleting or accumulating intermediates excessively.
2. Example: Citric Acid Cycle:
Overview: The citric acid cycle (Krebs cycle) is a key metabolic pathway that generates ATP,
NADH, and FADH2 through the oxidation of acetyl-CoA. It involves several intermediates,
such as citrate, alpha-ketoglutarate, and oxaloacetate.
Equilibrium: In the citric acid cycle, the concentrations of intermediates remain relatively
constant due to the cycle's continuous operation and the cell's regulatory mechanisms.
Energy Production: The cycle produces high-energy molecules (NADH, FADH2) that are used
in the electron transport chain to generate ATP, highlighting its role in cellular energy
metabolism.
3. Regulatory Mechanisms:
Allosteric Regulation: Key enzymes of the citric acid cycle, such as citrate synthase, isocitrate
dehydrogenase, and alpha-ketoglutarate dehydrogenase, are regulated by allosteric effectors
like ATP, ADP, NADH, and acetyl-CoA. These effectors either activate or inhibit enzyme
activity based on the cell's energy status.
Feedback Inhibition: Products of the citric acid cycle, such as NADH and ATP, inhibit earlier
steps in the cycle and other metabolic pathways to prevent the overproduction of intermediates
and ensure a balanced supply of metabolic substrates.
Conclusion:
Biochemical cycles in equilibrium are essential for sustaining life, as they ensure the consistent
production of energy and biomolecules needed for cellular processes. The citric acid cycle
exemplifies how dynamic equilibrium maintains metabolic balance, supports energy
production, and adapts to the cell's varying demands through precise regulatory mechanisms.

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Kinetic Proofreading in Biological Systems
Introduction:
Kinetic proofreading is a mechanism that enhances the accuracy of molecular recognition
processes, such as DNA replication, transcription, and translation. This concept, introduced by
John Hopfield and Jacques Ninio in the 1970s, is fundamental to understanding how cells
achieve high fidelity in biochemical reactions. By incorporating time delays and additional
energy consumption, kinetic proofreading allows biological systems to correct errors and
ensure precise molecular interactions.
1. Concept of Kinetic Proofreading:
Mechanism: Kinetic proofreading involves multiple checkpoints that allow the system to reject
incorrect substrates before a final product is formed. This increases the specificity of molecular
interactions, significantly reducing error rates.
Energy and Time Trade-off: The process consumes additional energy and time, which provides
the necessary checkpoints to distinguish between correct and incorrect substrates. This trade-
off is crucial for achieving high accuracy in biological processes.
2. DNA Replication:
Role in Fidelity: During DNA replication, DNA polymerases use kinetic proofreading to ensure
that only correctly paired nucleotides are incorporated into the growing DNA strand. The
polymerase stalls upon detecting a mismatch, allowing exonuclease activity to remove the
incorrect nucleotide before synthesis resumes.
Impact on Genomic Stability: By reducing the error rate during replication, kinetic
proofreading helps maintain genomic stability and prevent mutations that could lead to diseases
such as cancer.
3. Transcription and Translation:
Transcription: In transcription, RNA polymerase exhibits proofreading functions to correct
errors in RNA synthesis. The enzyme can backtrack to excise misincorporated nucleotides,
ensuring the production of accurate mRNA transcripts.
Translation: In translation, ribosomes use kinetic proofreading to ensure the correct aminoacyl-
tRNA is selected for peptide bond formation. This involves the initial selection of tRNA by the
ribosome and a subsequent proofreading step that verifies the correct codon-anticodon pairing
before peptide bond formation.
Accuracy in Protein Synthesis: The accuracy of translation is critical for producing functional
proteins and avoiding harmful mutations that can affect cellular function and organismal health.
4. Mathematical Models:
Overview: Mathematical models of kinetic proofreading describe the increase in accuracy
achieved through additional energy expenditure and time delays. These models help explain
the balance between speed and accuracy in biological systems.

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Hopfield and Ninio Models: The original models proposed by Hopfield and Ninio illustrate
how kinetic proofreading mechanisms can dramatically reduce error rates in molecular
recognition processes, providing a theoretical framework for understanding these phenomena.
Biological (living) systems are open; they generally reach a non-equilibrium steady state
(NESS). For theoretical studies, we take some reacting species concentrations as constants
under what is known as the chemiostatic condition [20]. The biochemical reactions inside cells
often involve low copy numbers of the reacting species. That is why, one models the kinetics
as stochastic, i.e., instead of concentrations use probabilities.
Extensions and Applications: Further research has extended these models to various biological
systems, offering insights into the design of synthetic biological circuits and the development
of novel therapeutic approaches.
5. Biological Implications:
Genetic Stability: Kinetic proofreading is crucial for maintaining genetic stability and
preventing diseases caused by mutations. Defects in proofreading mechanisms can lead to
increased mutation rates and a higher risk of genetic disorders.
Evolutionary Significance: The evolution of kinetic proofreading mechanisms reflects the
selective pressure to enhance the fidelity of genetic information transfer, ensuring the survival
and adaptation of organisms in diverse environments.
6. Applications in Biotechnology:
Genetic Engineering: Insights from kinetic proofreading are applied in biotechnology to
improve the accuracy of genetic engineering and molecular diagnostics. For example,
engineered polymerases with enhanced proofreading capabilities are used in high-fidelity DNA
synthesis and sequencing technologies.
Therapeutic Strategies: Understanding kinetic proofreading mechanisms has implications for
developing therapeutic strategies to correct genetic errors. This includes designing drugs that
target defective proofreading enzymes or enhancing the fidelity of gene editing techniques.
Summary:
Kinetic proofreading is a vital mechanism that ensures high fidelity in biological systems. The
mechanism satisfactorily justifies the advantage of non-equilibrium steady state condition
sustained by ATP-hydrolysis to achieve such high accuracy [21]. Its role in DNA replication,
transcription, and translation underscores its importance in maintaining genetic integrity and
cellular function. The study of kinetic proofreading has far-reaching implications for
understanding the molecular basis of genetic stability, evolutionary biology, and the
development of advanced biotechnological applications. By leveraging the principles of kinetic
proofreading, scientists can understand and enhance the accuracy and efficiency of various
molecular processes, paving the way for innovations in medicine and biotechnology.

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Acknowledgment: I would like to thank all the faculties of the Department of Chemistry,
Acharya Jagadish Chandra Bose College, Kolkata for their continuous support. I am grateful
to Dr Kinshuk Banerjee, Dept. of Chemistry, Acharya Jagadish Chandra Bose College for
introducing me to the topic of this review. I am also thankful to the Principal, Acharya Jagadish
Chandra Bose College for allowing me to use the infrastructure of the college for preparation
of my review work.

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 17. Gromadski KB, Rodnina MV (2004) Kinetic determinants of high-fidelity tRNA
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