What molecular mechanism produces double-strand DNA breaks during CRISPR-Cas9 genome editing?

Cas9 endonuclease uses two catalytic nuclease domains (HNH and RuvC) guided by single-guide RNA (sgRNA) to cleave both strands 3 bp upstream of the PAM.

What molecular machinery executes nuclear pre-mRNA splicing, and what conserved dinucleotides mark intron boundaries?

The spliceosome (composed of snRNPs U1, U2, U4, U5, U6) recognizing conserved 5'-GU (donor) and 3'-AG (acceptor) splice junctions.

What three major post-transcriptional modifications convert pre-mRNA into mature mRNA in eukaryotic nuclei?

1. Addition of a 5' 7-methylguanosine cap. 2. Splicing out of introns and ligating exons. 3. Addition of a 3' polyadenylation tail (~200–250 adenines).

In agarose gel electrophoresis, what physical property causes nucleic acids to migrate toward the positive anode?

The negatively charged phosphate backbone of nucleic acids, causing migration toward the positive electrode with smaller fragments moving faster.

How do histone acetyltransferases (HATs) and histone deacetylases (HDACs) oppositely regulate chromatin accessibility?

HATs add acetyl groups to basic lysine residues, neutralizing positive charges to relax chromatin (euchromatin) and activate transcription; HDACs remove acetyl groups, promoting condensation (heterochromatin) and repression.

What intermediate structural formation is produced during spliceosome-mediated intron excision?

A branched lariat structure formed by a 2'-5' phosphodiester linkage between the 5' splice donor guanosine and the 2'-OH of the branch-point adenosine.

What is meant by the statement that the universal genetic code is 'degenerate' (redundant)?

Multiple distinct three-nucleotide mRNA codons can specify the exact same amino acid (e.g., 6 different codons code for leucine).

How does DNA methylation at cytosine-guanine (CpG) islands affect eukaryotic gene transcription?

Methylation of cytosines to 5-methylcytosine recruits methyl-CpG-binding proteins and histone deacetylases, compacting chromatin and repressing (silencing) transcription.

What bacterial mRNA sequence aligns the small 30S ribosomal subunit to the start codon during translation initiation?

The Shine-Dalgarno sequence (AGGAGG), which base-pairs with the 3' end of the 16S rRNA.

What modified nucleotides are utilized in classic Sanger dideoxy sequencing to cause chain termination?

Dideoxynucleotide triphosphates (ddNTPs), which lack both the 2'-OH and 3'-OH groups required for phosphodiester bond formation.

What three stop codons signal translation termination, and what binds to them in place of a tRNA?

UAA, UAG, and UGA; protein release factors (RFs) bind to the A site and trigger hydrolytic cleavage of the ester bond linking the polypeptide to tRNA.

What multi-protein effector complex uses single-stranded guide miRNAs/siRNAs to target mRNA for degradation or translational repression?

RISC (RNA-Induced Silencing Complex), containing an Argonaute (Ago) catalytic family protein.

Why is Taq polymerase from Thermus aquaticus used in automated PCR thermal cycling instead of human DNA polymerase?

Taq is thermostable and survives repeated exposure to 95°C denaturation temperatures without denaturing or losing enzymatic activity.

How does glucose concentration regulate lac operon transcription via catabolite repression?

Low glucose triggers adenylate cyclase to produce cyclic AMP (cAMP); cAMP binds CAP/CRP, and the CAP-cAMP complex binds upstream of the promoter to recruit RNA polymerase for high-level transcription.

In CRISPR-Cas9 bacterial adaptive immunity and genome editing, what is the role of the Protospacer Adjacent Motif (PAM)?

A short conserved DNA sequence (5'-NGG-3' for SpCas9) directly adjacent to the target site, required for Cas9 binding and cleavage.

What is the RNA interference (RNAi) pathway, and which endoribonuclease initially processes pre-miRNA into short duplexes?

A post-transcriptional gene silencing mechanism where Dicer cleaves double-stranded precursor RNAs into ~21–23 nucleotide miRNAs or siRNAs.

What are the three temperature phases in a typical cycle of the Polymerase Chain Reaction (PCR)?

1. Denaturation (~94–96°C) to separate double-stranded target DNA. 2. Annealing (~50–65°C) for primers to bind complementary sequences. 3. Extension (~72°C) where thermostable Taq polymerase synthesizes new DNA.

What is the molecular mechanism of attenuation in the trp operon under high tryptophan conditions?

Rapid translation of the leader peptide causes ribosome progression over Trp codons, allowing formation of the 3-4 terminator hairpin loop that prematurely halts transcription.

Which subunit of bacterial RNA polymerase holoenzyme recognizes promoter elements (-10 Pribnow and -35 boxes)?

The sigma factor (e.g., sigma-70).

What is the Central Dogma of Molecular Biology, and what notable enzyme constitutes an exception to its classical flow?

DNA is transcribed into RNA, which is translated into protein; reverse transcriptase (present in retroviruses) synthesizes complementary DNA (cDNA) from an RNA molecule.

What core promoter sequence in eukaryotes is bound by the TATA-binding protein (TBP) subunit of TFIID?

The TATA box, located approximately 25 to 30 base pairs upstream of the transcription start site (+1).

What does Francis Crick's Wobble Hypothesis explain regarding codon-anticodon pairing at the third position?

Non-Watson-Crick base pairing is tolerated at the 3' codon / 5' anticodon wobble position (e.g., inosine pairing with U, C, or A), enabling fewer tRNAs to decode 61 sense codons.

How does the tryptophan (trp) operon switch off in the presence of abundant intracellular tryptophan?

Tryptophan acts as a corepressor, binding the inactive TrpR aporepressor to form an active complex that binds the operator and blocks transcription.

What is the structural difference between euchromatin and heterochromatin, and how does it correlate with transcriptional activity?

Euchromatin is decondensed, accessible, and transcriptionally active; heterochromatin is densely packed, inaccessible, and transcriptionally silent.

What small regulatory protein marks damaged or short-lived proteins for degradation by the 26S proteasome?

Ubiquitin, covalently attached to lysine residues via an E1-E2-E3 enzymatic cascade.

How does alternative splicing expand the functional proteome of an organism without increasing total gene number?

By selectively including or excluding different combinations of exons from a single pre-mRNA, generating multiple distinct functional protein isoforms.

Which eukaryotic RNA polymerases synthesize ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA) respectively?

RNA Polymerase I synthesizes major rRNAs (28S, 18S, 5.8S); RNA Polymerase II synthesizes mRNA and snRNAs; RNA Polymerase III synthesizes tRNA and 5S rRNA.

What type of nucleotide sequence is recognized by Type II restriction endonucleases (e.g., EcoRI)?

Palindromic sequences (e.g., 5'-GAATTC-3' on one strand and 5'-GAATTC-3' on the complementary antiparallel strand).

What are enhancers in eukaryotic gene regulation, and how do they function across thousands of base pairs?

Distal cis-regulatory DNA sequences bound by activator transcription factors that loop DNA with mediator complex assistance to contact the basal promoter.

How does prokaryotic Rho-independent (intrinsic) transcription termination work mechanically?

RNA polymerase transcribes a GC-rich inverted repeat that forms a stable hairpin stem-loop followed by a poly-uracil (poly-U) tract, destabilizing the weak rU-dA hybrid and causing transcript release.

What ribosomal enzyme catalyzes peptide bond formation during translation, and what is its biochemical nature?

Peptidyl transferase, which is a ribozyme activity intrinsic to the large ribosomal subunit RNA (23S rRNA in prokaryotes, 28S in eukaryotes).

In the bacterial lac operon, what molecules act as the repressor and the physiological inducer?

The LacI repressor protein binds the operator; allolactose (a lactose isomer formed by beta-galactosidase) acts as the inducer by binding LacI to release it.

Immunology (Advanced 1): Molecular Biology & Gene Regulation

Transcription, RNA processing, translation, operons, eukaryotic epigenetic regulation, and recombinant DNA technology

Level: advanced
Languages: en → en
Cards: 32
Markdown: Immunology (Advanced 1): Molecular Biology & Gene Regulation (.md)

Flashcards (32)

#	Front (Term / Question)	Back (Definition / Answer)
1	What molecular mechanism produces double-strand DNA breaks during CRISPR-Cas9 genome editing?	Cas9 endonuclease uses two catalytic nuclease domains (HNH and RuvC) guided by single-guide RNA (sgRNA) to cleave both strands 3 bp upstream of the PAM.
2	What molecular machinery executes nuclear pre-mRNA splicing, and what conserved dinucleotides mark intron boundaries?	The spliceosome (composed of snRNPs U1, U2, U4, U5, U6) recognizing conserved 5'-GU (donor) and 3'-AG (acceptor) splice junctions.
3	What three major post-transcriptional modifications convert pre-mRNA into mature mRNA in eukaryotic nuclei?	1. Addition of a 5' 7-methylguanosine cap. 2. Splicing out of introns and ligating exons. 3. Addition of a 3' polyadenylation tail (~200–250 adenines).
4	In agarose gel electrophoresis, what physical property causes nucleic acids to migrate toward the positive anode?	The negatively charged phosphate backbone of nucleic acids, causing migration toward the positive electrode with smaller fragments moving faster.
5	How do histone acetyltransferases (HATs) and histone deacetylases (HDACs) oppositely regulate chromatin accessibility?	HATs add acetyl groups to basic lysine residues, neutralizing positive charges to relax chromatin (euchromatin) and activate transcription; HDACs remove acetyl groups, promoting condensation (heterochromatin) and repression.
6	What intermediate structural formation is produced during spliceosome-mediated intron excision?	A branched lariat structure formed by a 2'-5' phosphodiester linkage between the 5' splice donor guanosine and the 2'-OH of the branch-point adenosine.
7	What is meant by the statement that the universal genetic code is 'degenerate' (redundant)?	Multiple distinct three-nucleotide mRNA codons can specify the exact same amino acid (e.g., 6 different codons code for leucine).
8	How does DNA methylation at cytosine-guanine (CpG) islands affect eukaryotic gene transcription?	Methylation of cytosines to 5-methylcytosine recruits methyl-CpG-binding proteins and histone deacetylases, compacting chromatin and repressing (silencing) transcription.
9	What bacterial mRNA sequence aligns the small 30S ribosomal subunit to the start codon during translation initiation?	The Shine-Dalgarno sequence (AGGAGG), which base-pairs with the 3' end of the 16S rRNA.
10	What modified nucleotides are utilized in classic Sanger dideoxy sequencing to cause chain termination?	Dideoxynucleotide triphosphates (ddNTPs), which lack both the 2'-OH and 3'-OH groups required for phosphodiester bond formation.
11	What three stop codons signal translation termination, and what binds to them in place of a tRNA?	UAA, UAG, and UGA; protein release factors (RFs) bind to the A site and trigger hydrolytic cleavage of the ester bond linking the polypeptide to tRNA.
12	What multi-protein effector complex uses single-stranded guide miRNAs/siRNAs to target mRNA for degradation or translational repression?	RISC (RNA-Induced Silencing Complex), containing an Argonaute (Ago) catalytic family protein.
13	Why is Taq polymerase from Thermus aquaticus used in automated PCR thermal cycling instead of human DNA polymerase?	Taq is thermostable and survives repeated exposure to 95°C denaturation temperatures without denaturing or losing enzymatic activity.
14	How does glucose concentration regulate lac operon transcription via catabolite repression?	Low glucose triggers adenylate cyclase to produce cyclic AMP (cAMP); cAMP binds CAP/CRP, and the CAP-cAMP complex binds upstream of the promoter to recruit RNA polymerase for high-level transcription.
15	In CRISPR-Cas9 bacterial adaptive immunity and genome editing, what is the role of the Protospacer Adjacent Motif (PAM)?	A short conserved DNA sequence (5'-NGG-3' for SpCas9) directly adjacent to the target site, required for Cas9 binding and cleavage.
16	What is the RNA interference (RNAi) pathway, and which endoribonuclease initially processes pre-miRNA into short duplexes?	A post-transcriptional gene silencing mechanism where Dicer cleaves double-stranded precursor RNAs into ~21–23 nucleotide miRNAs or siRNAs.
17	What are the three temperature phases in a typical cycle of the Polymerase Chain Reaction (PCR)?	1. Denaturation (~94–96°C) to separate double-stranded target DNA. 2. Annealing (~50–65°C) for primers to bind complementary sequences. 3. Extension (~72°C) where thermostable Taq polymerase synthesizes new DNA.
18	What is the molecular mechanism of attenuation in the trp operon under high tryptophan conditions?	Rapid translation of the leader peptide causes ribosome progression over Trp codons, allowing formation of the 3-4 terminator hairpin loop that prematurely halts transcription.
19	Which subunit of bacterial RNA polymerase holoenzyme recognizes promoter elements (-10 Pribnow and -35 boxes)?	The sigma factor (e.g., sigma-70).
20	What is the Central Dogma of Molecular Biology, and what notable enzyme constitutes an exception to its classical flow?	DNA is transcribed into RNA, which is translated into protein; reverse transcriptase (present in retroviruses) synthesizes complementary DNA (cDNA) from an RNA molecule.
21	What core promoter sequence in eukaryotes is bound by the TATA-binding protein (TBP) subunit of TFIID?	The TATA box, located approximately 25 to 30 base pairs upstream of the transcription start site (+1).
22	What does Francis Crick's Wobble Hypothesis explain regarding codon-anticodon pairing at the third position?	Non-Watson-Crick base pairing is tolerated at the 3' codon / 5' anticodon wobble position (e.g., inosine pairing with U, C, or A), enabling fewer tRNAs to decode 61 sense codons.
23	How does the tryptophan (trp) operon switch off in the presence of abundant intracellular tryptophan?	Tryptophan acts as a corepressor, binding the inactive TrpR aporepressor to form an active complex that binds the operator and blocks transcription.
24	What is the structural difference between euchromatin and heterochromatin, and how does it correlate with transcriptional activity?	Euchromatin is decondensed, accessible, and transcriptionally active; heterochromatin is densely packed, inaccessible, and transcriptionally silent.
25	What small regulatory protein marks damaged or short-lived proteins for degradation by the 26S proteasome?	Ubiquitin, covalently attached to lysine residues via an E1-E2-E3 enzymatic cascade.
26	How does alternative splicing expand the functional proteome of an organism without increasing total gene number?	By selectively including or excluding different combinations of exons from a single pre-mRNA, generating multiple distinct functional protein isoforms.
27	Which eukaryotic RNA polymerases synthesize ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA) respectively?	RNA Polymerase I synthesizes major rRNAs (28S, 18S, 5.8S); RNA Polymerase II synthesizes mRNA and snRNAs; RNA Polymerase III synthesizes tRNA and 5S rRNA.
28	What type of nucleotide sequence is recognized by Type II restriction endonucleases (e.g., EcoRI)?	Palindromic sequences (e.g., 5'-GAATTC-3' on one strand and 5'-GAATTC-3' on the complementary antiparallel strand).
29	What are enhancers in eukaryotic gene regulation, and how do they function across thousands of base pairs?	Distal cis-regulatory DNA sequences bound by activator transcription factors that loop DNA with mediator complex assistance to contact the basal promoter.
30	How does prokaryotic Rho-independent (intrinsic) transcription termination work mechanically?	RNA polymerase transcribes a GC-rich inverted repeat that forms a stable hairpin stem-loop followed by a poly-uracil (poly-U) tract, destabilizing the weak rU-dA hybrid and causing transcript release.
31	What ribosomal enzyme catalyzes peptide bond formation during translation, and what is its biochemical nature?	Peptidyl transferase, which is a ribozyme activity intrinsic to the large ribosomal subunit RNA (23S rRNA in prokaryotes, 28S in eukaryotes).
32	In the bacterial lac operon, what molecules act as the repressor and the physiological inducer?	The LacI repressor protein binds the operator; allolactose (a lactose isomer formed by beta-galactosidase) acts as the inducer by binding LacI to release it.