Question 1

What is the structure of DNA and who discovered it?

Accepted Answer

DNA is a double helix structure consisting of two antiparallel strands of nucleotides connected by hydrogen bonds between complementary base pairs (A-T with 2 bonds, G-C with 3 bonds). James Watson and Francis Crick proposed this model in 1953, building on X-ray crystallography data from Rosalind Franklin. The sugar-phosphate backbone runs on the outside while the bases face inward, creating major and minor grooves important for protein binding.

Question 2

What are the key differences between DNA and RNA?

Accepted Answer

DNA contains deoxyribose sugar while RNA contains ribose (with an extra hydroxyl group at 2' position), making RNA less stable. DNA uses thymine (T) as a base while RNA uses uracil (U). DNA is typically double-stranded and serves as long-term genetic storage, while RNA is usually single-stranded and functions in protein synthesis, gene regulation, and catalysis. DNA is found primarily in the nucleus, while RNA is synthesized in the nucleus and functions largely in the cytoplasm.

Question 3

What is the central dogma of molecular biology?

Accepted Answer

The central dogma describes the flow of genetic information in cells: DNA is transcribed into RNA, which is then translated into protein (DNA -> RNA -> Protein). Francis Crick proposed this concept in 1958. While this represents the primary information flow, exceptions exist such as reverse transcription (RNA to DNA in retroviruses) and RNA replication in RNA viruses. The dogma emphasizes that information flows from nucleic acids to proteins but not in reverse.

Question 4

What is transcription and what enzyme catalyzes it?

Accepted Answer

Transcription is the process of synthesizing RNA from a DNA template, representing the first step in gene expression. RNA polymerase is the enzyme that catalyzes transcription by reading the template strand 3' to 5' and synthesizing RNA 5' to 3'. In prokaryotes, a single RNA polymerase handles all transcription, while eukaryotes have RNA Pol I (rRNA), RNA Pol II (mRNA), and RNA Pol III (tRNA and small RNAs). The process involves initiation at promoters, elongation, and termination.

Question 5

What is translation and where does it occur?

Accepted Answer

Translation is the process of synthesizing proteins from mRNA, occurring on ribosomes in the cytoplasm. The ribosome reads the mRNA sequence in codons (three nucleotides) and matches them with complementary anticodons on tRNA molecules carrying specific amino acids. Translation proceeds through initiation (ribosome assembly at start codon AUG), elongation (amino acid chain growth), and termination (at stop codons UAA, UAG, UGA). The process requires mRNA, tRNA, ribosomes, and various protein factors.

Question 6

What are codons and how many amino acids do they encode?

Accepted Answer

Codons are three-nucleotide sequences in mRNA that specify particular amino acids during translation. With 4 nucleotides possible at each position, there are 64 possible codons (4^3). These encode 20 standard amino acids plus 3 stop signals (UAA, UAG, UGA), making the genetic code degenerate (redundant) - multiple codons can specify the same amino acid. AUG serves as both the start codon and codes for methionine. The code is nearly universal across all organisms with minor variations in mitochondria and some organisms.

Question 7

What is semiconservative DNA replication?

Accepted Answer

Semiconservative replication means that each new DNA molecule consists of one original (parental) strand and one newly synthesized strand. This was demonstrated by Meselson and Stahl in 1958 using density gradient centrifugation with heavy nitrogen isotopes. During replication, the double helix unwinds at origins of replication, and each strand serves as a template for synthesizing a complementary strand. This mechanism ensures accurate transmission of genetic information and was predicted by Watson and Crick based on DNA structure.

Question 8

What is the role of primers in DNA replication?

Accepted Answer

Primers are short RNA sequences (about 10 nucleotides) synthesized by primase that provide a free 3'-OH group for DNA polymerase to begin synthesis. DNA polymerase cannot initiate new strand synthesis de novo and can only add nucleotides to an existing 3' end. On the leading strand, only one primer is needed, while the lagging strand requires multiple primers for each Okazaki fragment. After DNA synthesis, primers are removed by RNase H or DNA polymerase I and replaced with DNA by repair mechanisms.

Question 9

What post-transcriptional modifications occur to eukaryotic mRNA?

Accepted Answer

Eukaryotic pre-mRNA undergoes three major modifications: 5' capping (addition of 7-methylguanosine cap that protects from degradation and aids ribosome binding), 3' polyadenylation (addition of poly-A tail of 100-250 adenines for stability and export), and splicing (removal of introns and joining of exons by the spliceosome). These modifications occur in the nucleus before export to the cytoplasm and are essential for mRNA stability, nuclear export, and efficient translation.

Question 10

What is the difference between introns and exons?

Accepted Answer

Exons are the coding sequences of a gene that are retained in mature mRNA and translated into protein, while introns are intervening non-coding sequences that are removed during RNA splicing. Eukaryotic genes typically contain multiple introns and exons, with introns often much longer than exons. Alternative splicing allows different combinations of exons to produce multiple protein isoforms from a single gene. Prokaryotes generally lack introns, though some are found in archaeal and bacterial genes.

Question 11

What is a promoter and what is its function?

Accepted Answer

A promoter is a DNA sequence located upstream (5') of a gene that serves as the binding site for RNA polymerase and transcription factors to initiate transcription. In prokaryotes, common promoter elements include the -10 box (TATAAT, Pribnow box) and -35 box recognized by sigma factor. Eukaryotic promoters contain elements like the TATA box, initiator element, and various regulatory sequences. The strength of a promoter determines the frequency of transcription initiation and thus gene expression levels.

Question 12

What is gel electrophoresis and how does it work?

Accepted Answer

Gel electrophoresis is a technique for separating DNA, RNA, or proteins based on size and charge by applying an electric field across a gel matrix (agarose or polyacrylamide). Nucleic acids, being negatively charged due to phosphate groups, migrate toward the positive electrode (anode). Smaller molecules move faster through the gel pores, resulting in size-based separation. DNA is visualized using fluorescent dyes like ethidium bromide or SYBR Safe, and size is determined by comparison to molecular weight markers.

Question 13

What are restriction enzymes and how are they used?

Accepted Answer

Restriction enzymes (restriction endonucleases) are bacterial enzymes that cut DNA at specific recognition sequences, typically 4-8 base pairs long. They serve as a bacterial defense mechanism against viral DNA. Type II restriction enzymes, most commonly used in molecular biology, cut at or near their recognition sites producing either blunt ends or sticky (cohesive) ends with overhangs. They are essential tools for DNA cloning, mapping, and genetic engineering, allowing precise cutting and recombination of DNA fragments.

Question 14

What is DNA ligase and what is its function?

Accepted Answer

DNA ligase is an enzyme that catalyzes the formation of phosphodiester bonds between the 3'-OH and 5'-phosphate ends of DNA strands, joining DNA fragments together. It is essential for DNA replication (joining Okazaki fragments), DNA repair, and recombinant DNA technology. T4 DNA ligase, derived from bacteriophage T4, is commonly used in molecular cloning to ligate DNA inserts into vectors. The enzyme requires ATP (or NAD+ in bacteria) as a cofactor for the ligation reaction.

Question 15

What is an operon and how does the lac operon work?

Accepted Answer

An operon is a cluster of genes under control of a single promoter, common in prokaryotes, that are transcribed together as a polycistronic mRNA. The lac operon in E. coli contains genes for lactose metabolism (lacZ, lacY, lacA) and is regulated by a repressor protein and CAP activator. In the absence of lactose, the repressor binds the operator blocking transcription. When lactose is present, it is converted to allolactose which binds and inactivates the repressor, allowing transcription. This represents negative inducible regulation.

Question 16

Explain the role of transcription factors in eukaryotic gene expression.

Accepted Answer

Eukaryotic transcription factors (TFs) are proteins that regulate gene expression by binding to specific DNA sequences. General TFs (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH) are required for basal transcription from all promoters and assemble with RNA Pol II at the promoter. Specific TFs (activators and repressors) bind enhancers, silencers, or response elements to modulate transcription rates. They work through recruitment of coactivators, chromatin remodelers, or the mediator complex. Combinatorial control by multiple TFs allows precise spatial and temporal gene regulation.

Question 17

How does chromatin structure affect gene expression?

Accepted Answer

Chromatin structure regulates gene accessibility. Tightly packed heterochromatin is transcriptionally silent, while open euchromatin allows active transcription. Histone modifications play key roles: acetylation (by HATs) generally activates genes by loosening chromatin, while deacetylation (by HDACs) represses. Methylation effects depend on location - H3K4me3 activates, H3K27me3 represses. Chromatin remodeling complexes (SWI/SNF, ISWI) use ATP to reposition nucleosomes, exposing or hiding regulatory sequences. DNA methylation at CpG islands typically silences genes. This epigenetic regulation is heritable and crucial for development and disease.

Question 18

Describe the key enzymes involved in DNA replication and their functions.

Accepted Answer

DNA replication requires multiple enzymes working in coordination. Helicase unwinds the double helix at replication forks. Single-strand binding proteins (SSBs) stabilize unwound strands. Primase synthesizes RNA primers. DNA polymerase III is the main replicative enzyme, synthesizing DNA 5' to 3' with proofreading ability. DNA polymerase I removes RNA primers and fills gaps. DNA ligase seals nicks between Okazaki fragments. Topoisomerases relieve supercoiling ahead of the fork. Sliding clamp (PCNA in eukaryotes) increases processivity. This coordinated enzyme system ensures rapid, accurate replication.

Question 19

Explain the mechanism of RNA interference (RNAi).

Accepted Answer

RNA interference is a gene silencing mechanism triggered by double-stranded RNA. The enzyme Dicer cleaves dsRNA into small interfering RNAs (siRNAs) of 21-23 nucleotides. These siRNAs are loaded into the RNA-induced silencing complex (RISC), where Argonaute proteins unwind the duplex and retain the guide strand. The guide strand directs RISC to complementary mRNA targets, leading to mRNA cleavage or translational repression. MicroRNAs (miRNAs) work similarly but typically cause translational inhibition through partial complementarity. RNAi is used for gene knockdown studies and has therapeutic potential.

Question 20

What are molecular chaperones and how do they assist protein folding?

Accepted Answer

Molecular chaperones are proteins that assist in the proper folding of other proteins without being part of the final structure. The Hsp70 family binds to hydrophobic regions of nascent polypeptides, preventing aggregation and allowing proper folding. Chaperonins (like GroEL/GroES in bacteria, TRiC in eukaryotes) provide isolated chambers where proteins can fold without interference. Hsp90 assists in the maturation of signaling proteins. Chaperones use ATP hydrolysis to drive conformational changes. Misfolding despite chaperone assistance can lead to diseases like Alzheimer's and Parkinson's.

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What is the structure of DNA and who discovered it?

What are the key differences between DNA and RNA?

What is the central dogma of molecular biology?

What is transcription and what enzyme catalyzes it?

What is translation and where does it occur?

What are codons and how many amino acids do they encode?

What is semiconservative DNA replication?

What is the role of primers in DNA replication?

What post-transcriptional modifications occur to eukaryotic mRNA?

What is the difference between introns and exons?

What is a promoter and what is its function?

What is gel electrophoresis and how does it work?

What are restriction enzymes and how are they used?

What is DNA ligase and what is its function?

What is an operon and how does the lac operon work?

Explain the role of transcription factors in eukaryotic gene expression.

How does chromatin structure affect gene expression?

Describe the key enzymes involved in DNA replication and their functions.

Explain the mechanism of RNA interference (RNAi).

What are molecular chaperones and how do they assist protein folding?

What is alternative splicing and why is it significant?

What are the major DNA repair mechanisms?

What are telomeres and what is the role of telomerase?

Describe the structure and function of ribosomes.

Explain the basic components of cell signal transduction pathways.

What are the differences between Southern, Northern, and Western blotting?

How does real-time PCR (qPCR) enable quantification of nucleic acids?

What are common post-translational modifications and their functions?

Explain the MAPK signaling cascade and its biological significance.

What are reporter genes and how are they used in research?

How is mRNA stability regulated in cells?

What are cell cycle checkpoints and how do they function?

How does chromatin immunoprecipitation (ChIP) work?

Explain bacterial two-component signaling systems.

What factors affect nucleic acid hybridization stringency?

How do you analyze and minimize CRISPR off-target effects in gene editing experiments?

Explain the workflow and analytical challenges in single-cell RNA sequencing.

How do you design and interpret epigenome-wide association studies (EWAS)?

How has AlphaFold changed protein structure prediction and what are its limitations?

What are the advantages of long-read sequencing and how do you analyze such data?

How does ribosome profiling work and what insights does it provide?

How does liquid-liquid phase separation contribute to gene regulation?

How do you design and optimize synthetic gene circuits?

How do you study 3D genome organization and its functional implications?

How does cryo-EM enable structure determination and what are the key considerations?

How do you integrate multi-omics data to understand biological systems?

How is optogenetics used to study cell signaling dynamics?

Compare base editing and prime editing with traditional CRISPR-Cas9.

How does proteostasis network dysfunction contribute to disease?

How does spatial transcriptomics provide insights beyond single-cell RNA-seq?

What is recombinant DNA technology?

What is PCR and what are its basic components?

What is molecular cloning and what are its basic steps?

What are the essential components of a plasmid cloning vector?

What is CRISPR-Cas9 and how does it work?

What are the common methods for bacterial transformation?

What is blue-white screening and how does it work?

What is the difference between sticky ends and blunt ends in DNA cloning?

What is reverse transcription and when is it used?

What are the different types of cloning vectors and their capacities?

What is the difference between transfection and transduction?

What features distinguish expression vectors from cloning vectors?

What are the basic principles of PCR primer design?

What is Gateway cloning and what are its advantages?

What is site-directed mutagenesis and how is it performed?

How does Gibson assembly work and when is it preferred over traditional cloning?

What factors should be considered when designing CRISPR guide RNAs?

How do you optimize a multiplex PCR reaction?

Describe the process of lentiviral vector production for gene delivery.