CSIR NET Life Sciences Fundamental Processes Syllabus

Outline base excision repair (BER).

A DNA glycosylase removes a damaged base creating an AP site; AP endonuclease nicks the backbone; the gap is filled by DNA polymerase and sealed by ligase.

Outline nucleotide excision repair (NER).

Bulky helix-distorting lesions (e.g., pyrimidine dimers) are recognized; the damaged strand is incised on both sides (UvrABC in E. coli), the oligonucleotide removed, and the gap filled and sealed.

What human disease results from defective nucleotide excision repair?

Xeroderma pigmentosum (XP), causing extreme UV sensitivity and high skin cancer risk.

What is the SOS response in E. coli?

An inducible DNA-damage response controlled by RecA and LexA; RecA stimulates LexA autocleavage, derepressing SOS genes including error-prone translesion polymerases.

What is translesion synthesis (TLS)?

Specialized error-prone DNA polymerases (e.g., Pol IV, Pol V in E. coli) replicate across damaged templates, allowing replication to continue at the cost of higher mutation rate.

What is homologous recombination?

Exchange of genetic information between two homologous DNA molecules; used in repair of double-strand breaks and during meiosis to generate diversity.

What is the role of RecA in homologous recombination?

RecA coats single-stranded DNA forming a nucleoprotein filament that promotes strand invasion and homology search/pairing with a duplex (eukaryotic homolog: Rad51).

What is a Holliday junction?

A four-stranded cross-shaped DNA intermediate in homologous recombination formed by reciprocal strand exchange; it can branch migrate and be resolved into recombinant or non-recombinant products.

What is the function of the RecBCD complex in E. coli recombination?

It is a helicase/nuclease that unwinds DNA from a double-strand break, recognizes Chi sites, and generates 3' single-stranded ends for RecA loading.

How does site-specific recombination differ from homologous recombination?

Site-specific recombination occurs between short specific DNA sequences (no extensive homology needed) and is mediated by specific recombinases; homologous recombination requires extensive sequence homology.

Give the classic example of site-specific recombination by phage lambda.

Integration of phage λ into the E. coli chromosome at attP/attB sites, catalyzed by lambda integrase (Int) plus IHF, forming the prophage.

What are the two major families of site-specific recombinases?

The tyrosine recombinases (e.g., λ integrase, Cre, FLP) and the serine recombinases (e.g., resolvases, invertases like Hin).

What is a promoter in prokaryotic transcription, and name its consensus elements?

The DNA sequence where RNA polymerase binds to start transcription; in E. coli it includes the -10 (Pribnow box, TATAAT) and -35 (TTGACA) consensus elements.

What is the role of the sigma (σ) factor in bacterial transcription?

Sigma factor confers promoter recognition specificity to RNA polymerase, enabling initiation; it dissociates after initiation. Core enzyme + σ = holoenzyme.

What are the subunits of E. coli core RNA polymerase?

Two α, one β, one β', and one ω (α2ββ'ω); adding sigma (σ) gives the holoenzyme.

Name the three eukaryotic nuclear RNA polymerases and their products.

RNA Pol I makes rRNA (28S, 18S, 5.8S); RNA Pol II makes mRNA and most snRNAs; RNA Pol III makes tRNA, 5S rRNA, and small RNAs.

Which eukaryotic RNA polymerase is inhibited by α-amanitin?

RNA polymerase II is highly sensitive to α-amanitin; Pol III is moderately sensitive; Pol I is resistant.

What is the TATA box and which factor recognizes it?

A core promoter element (~ -25 to -30) in many Pol II genes; recognized by TBP (TATA-binding protein), a subunit of TFIID.

List the general transcription factors that assemble the Pol II pre-initiation complex.

TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH, which assemble with RNA Pol II at the promoter to form the initiation complex.

What enzymatic activities does TFIIH provide?

Helicase activity to melt promoter DNA (open complex) and a CTD kinase activity that phosphorylates the RNA Pol II C-terminal domain to promote promoter escape.

How do transcriptional activators and repressors differ?

Activators bind enhancers and recruit coactivators/general machinery to increase transcription; repressors bind silencers or compete/block to decrease transcription.

What modification caps the 5' end of eukaryotic mRNA and what is its function?

A 7-methylguanosine (m7G) cap added via a 5'-5' triphosphate linkage; it protects mRNA from degradation, aids splicing, export, and ribosome (cap) recognition for translation initiation.

What is rho-dependent transcription termination in bacteria?

Rho protein, an ATP-dependent helicase, binds the nascent RNA (rut site), translocates to the polymerase, and dissociates the RNA-DNA hybrid to terminate transcription.

What is rho-independent (intrinsic) termination?

Termination caused by a GC-rich hairpin in the RNA followed by a poly-U stretch, which destabilizes the RNA-DNA hybrid and releases the transcript without Rho.

What is the polyadenylation signal and the poly-A tail's function in eukaryotic mRNA?

The AAUAAA signal directs cleavage and addition of a poly-A tail (~200 A's); it enhances mRNA stability, export, and translation efficiency.

What is the role of spliceosome and snRNAs in RNA processing?

The spliceosome (snRNPs U1, U2, U4, U5, U6) removes introns and joins exons; it recognizes the 5' splice site (GU), branch point (A), and 3' splice site (AG).

What is alternative splicing?

Producing different mature mRNAs (and protein isoforms) from a single pre-mRNA by including/excluding different exons, increasing proteome diversity.

What does the wobble hypothesis explain?

That the third (5') base of the anticodon can pair non-standardly with the codon's third base, allowing one tRNA to read multiple codons and explaining codon degeneracy.

List the key features of the genetic code.

It is triplet, degenerate (redundant), non-overlapping, comma-less, nearly universal, and has start (AUG) and stop (UAA, UAG, UGA) codons.

What is the composition of the prokaryotic 70S ribosome?

A 30S small subunit (16S rRNA + 21 proteins) and a 50S large subunit (23S and 5S rRNA + ~34 proteins).

What is the composition of the eukaryotic 80S ribosome?

A 40S small subunit (18S rRNA + proteins) and a 60S large subunit (28S, 5.8S, and 5S rRNA + proteins).

Name the three ribosomal tRNA-binding sites and their roles.

A site (aminoacyl, accepts incoming tRNA), P site (peptidyl, holds growing chain), and E site (exit, releases deacylated tRNA).

What is the Shine-Dalgarno sequence?

A purine-rich ribosome-binding site upstream of the start codon in prokaryotic mRNA that base-pairs with 16S rRNA to position the ribosome for initiation.

What initiator tRNA and amino acid begin translation in bacteria versus eukaryotes?

Bacteria use fMet-tRNAf (N-formylmethionine); eukaryotes use Met-tRNAi (unformylated methionine).

Name the prokaryotic translation initiation factors and their roles.

IF1 (blocks A site), IF2 (a GTPase that delivers fMet-tRNA to the P site), and IF3 (prevents premature subunit joining / aids mRNA selection).

What is the role of EF-Tu and EF-G in bacterial elongation?

EF-Tu (GTP) delivers aminoacyl-tRNA to the A site; EF-G (GTP) catalyzes translocation, moving the ribosome one codon along the mRNA.

What catalyzes peptide bond formation during translation?

Peptidyl transferase activity, a ribozyme located in the large subunit's 23S rRNA (50S in prokaryotes).

How does translation terminate?

A stop codon in the A site is recognized by release factors (RF1/RF2 in bacteria; eRF1 in eukaryotes), which trigger hydrolysis and release of the polypeptide; RF3/eRF3 (GTPases) assist.

What is aminoacylation (charging) of tRNA?

Attachment of the correct amino acid to its tRNA's 3' CCA end by aminoacyl-tRNA synthetase, using ATP (forming aminoacyl-AMP intermediate), producing aminoacyl-tRNA.

What is tRNA identity?

The set of nucleotide features (identity elements, often in the anticodon and acceptor stem) that allow an aminoacyl-tRNA synthetase to recognize and charge the correct tRNA.

How does aminoacyl-tRNA synthetase perform translational proofreading?

Through editing (proofreading) activity that hydrolyzes incorrectly activated amino acids or mischarged tRNAs (pre-transfer and post-transfer editing), ensuring fidelity.

What are the two classes of aminoacyl-tRNA synthetases?

Class I (mostly monomeric, charge the 2'-OH of tRNA, approach the minor groove) and Class II (often dimeric, charge the 3'-OH, approach the major groove).

Name a translation inhibitor that blocks the prokaryotic 30S subunit and its effect.

Streptomycin binds the 30S subunit causing misreading and inhibiting initiation; tetracycline blocks aminoacyl-tRNA binding to the A site of the 30S.

How does chloramphenicol inhibit translation?

It binds the 50S subunit and inhibits peptidyl transferase activity, blocking peptide bond formation in prokaryotes.

How does puromycin inhibit translation?

It mimics aminoacyl-tRNA, enters the A site, accepts the growing chain, and causes premature chain termination/release; acts on both pro- and eukaryotes.

How does cycloheximide inhibit translation, and what does it spare?

It blocks the peptidyl transferase/translocation step of the eukaryotic 60S subunit; it does not affect prokaryotic ribosomes.

How does diphtheria toxin inhibit eukaryotic translation?

It ADP-ribosylates eEF2 (elongation factor 2), inactivating it and blocking translocation.

Give three examples of post-translational modifications of proteins.

Phosphorylation, glycosylation, and proteolytic cleavage; others include acetylation, methylation, ubiquitination, lipidation, and disulfide bond formation.

What is the function of the signal sequence in protein targeting?

An N-terminal hydrophobic sequence recognized by the signal recognition particle (SRP) that directs ribosomes to the ER for co-translational translocation of secretory/membrane proteins.

How does the lac operon function as a model of prokaryotic gene regulation?

In absence of lactose, the LacI repressor binds the operator blocking transcription; allolactose (inducer) inactivates the repressor (negative control); CAP-cAMP activates transcription when glucose is low (positive control).

What is catabolite repression (the glucose effect)?

When glucose is present, cAMP is low, CAP cannot bind, and operons like lac are poorly transcribed even with inducer, ensuring glucose is used preferentially.

How is the trp operon regulated by repression and attenuation?

High tryptophan activates the Trp repressor (corepressor = Trp) to block transcription; attenuation uses a leader RNA that forms a terminator hairpin when tryptophan/charged tRNA is abundant, prematurely halting transcription.

How does phage lambda choose between lytic and lysogenic cycles?

The cI repressor (maintains lysogeny by binding OR/OL) versus Cro protein (promotes lytic cycle) compete; their balance, influenced by host conditions, determines the genetic switch.

What is the role of chromatin remodeling in eukaryotic gene expression?

ATP-dependent remodeling complexes (e.g., SWI/SNF) reposition or evict nucleosomes to expose or occlude promoters, regulating transcription factor access.

How do histone acetylation and deacetylation affect gene expression?

Histone acetylation (by HATs) loosens chromatin and activates transcription; deacetylation (by HDACs) compacts chromatin and represses transcription.

How does DNA methylation contribute to gene silencing in eukaryotes?

Methylation of cytosines at CpG islands (by DNA methyltransferases) typically represses transcription by blocking factor binding and recruiting repressive complexes; basis of heterochromatin and imprinting.

What are the structural and functional differences between euchromatin and heterochromatin?

Euchromatin is loosely packed, gene-rich, and transcriptionally active; heterochromatin is condensed, gene-poor, generally transcriptionally silent, and often methylated/marked by H3K9me.

What is RNA interference (RNAi) and its role in gene silencing?

Small RNAs (siRNA/miRNA) loaded into RISC guide sequence-specific mRNA cleavage or translational repression, silencing gene expression post-transcriptionally; Dicer generates the small RNAs.