Exam Notes on Noncoding RNAs, Genome Defense, Gene Expression, Plasmids, Viruses, and Transposons

Noncoding RNAs

• Coding RNAs: Carry genetic messages from DNA to ribosomes for protein synthesis.
  • mRNA (messenger RNA): Contains codons read by ribosomes during translation.
• Noncoding RNAs (ncRNAs): RNAs transcribed from DNA but not translated; have various functional roles.
  • tRNA (transfer RNA): Brings amino acids to ribosomes during translation.
  • rRNA (ribosomal RNA): Structural and catalytic component of ribosomes.
  • snRNA (small nuclear RNA): Part of the spliceosome complex, removes introns from pre-mRNA.
  • snoRNA (small nucleolar RNA): Guide RNAs for modifying rRNA, mainly through methylation and pseudouridylation.
  • miRNA (microRNA) and siRNA (small interfering RNA): Involved in gene silencing and regulation.
  • lncRNA (long noncoding RNA): Functions include chromatin remodeling, transcriptional regulation, and scaffolding for protein complexes.
  • gRNA (guide RNA): Used in RNA editing, particularly in kinetoplastid mitochondria.
  • eRNA (enhancer RNA): Transcribed from enhancer regions; may facilitate enhancer-promoter interactions.
  • circRNA (circular RNA): Arise from back-splicing events; may regulate gene expression by sponging miRNAs or interacting with proteins.

Functions of Noncoding RNAs

• Ribozymes: RNAs that catalyze chemical reactions.
  • Example: Self-splicing introns, RNase P (cleaves precursor tRNA).
• Scaffolding: ncRNAs that provide a structural platform for protein complexes.
  • Example: NEAT1 in paraspeckles, HOTAIR in chromatin remodeling.
• Regulation: ncRNAs regulate gene expression at transcriptional or post-transcriptional levels.
  • miRNAs and siRNAs bind to mRNAs and inhibit translation or promote degradation.
• Defense: ncRNAs protect against foreign nucleic acids.
  • CRISPR RNAs (crRNAs) in prokaryotes recognize viral DNA; siRNAs can silence viral RNAs.

Prokaryotic Noncoding RNAs

• sRNAs:
  • Short RNAs (50-200 nt) that often bind to target mRNAs to affect stability or translation.
  • Can act by base-pairing with mRNAs, altering accessibility to ribosomes or RNases.
• tmRNA (transfer-messenger RNA):
  • Combines functions of tRNA and mRNA.
  • Rescues ribosomes stalled on damaged mRNAs.
  • Tags incomplete proteins for degradation via the protease system.
• CRISPR RNAs / Cas Proteins:
  • Part of a bacterial adaptive immune system.
  • crRNAs are derived from viral DNA inserts (spacers) and guide Cas proteins to matching DNA sequences for cleavage.
• 6S RNA:
  • Binds to RNA polymerase and mimics DNA to inhibit transcription during stationary phase.
  • Released when transcription resumes, allowing recovery from dormancy.

Eukaryotic Noncoding RNAs

• snoRNAs:
  • Found in the nucleolus.
  • Modify rRNA through 2’-O-methylation and pseudouridylation.
  • Guide enzymes to specific sites on rRNA via base pairing.
• snRNAs:
  • Involved in pre-mRNA splicing.
  • Components of the spliceosome (e.g., U1, U2, U4, U5, U6).
• gRNAs:
  • Guide RNA editing, especially in mitochondria of protozoa.
  • Direct insertion/deletion of uridines in mRNA.
• eRNAs (enhancer RNAs):
  • Transcribed from active enhancers.
  • Non-polyadenylated, short-lived RNAs.
  • May help recruit transcriptional machinery or maintain enhancer-promoter looping.
• Architectural RNAs:
  • Structural components of nuclear substructures (e.g., NEAT1 for paraspeckles).
  • Organize 3D genome architecture.
• Circular RNAs:
  • Formed by back-splicing of exons.
  • Can act as miRNA sponges, regulators of transcription, or even templates for translation in rare cases.
• siRNAs:
  • Arise from long dsRNA precursors.
  • Perfectly base-pair with target mRNAs → mRNA cleavage by RISC.
  • Part of RNA interference (RNAi).
• miRNAs:
  • Endogenously encoded.
  • Imperfect base-pairing with target mRNAs → translation repression or degradation.
  • Regulate gene expression post-transcriptionally.

Genome Defense: RNA Interference (RNAi)

• How it works:
  1. Double-stranded RNA (dsRNA) is recognized in the cell.
  2. Dicer, an RNase III enzyme, cleaves the dsRNA into ~21-23 nt siRNAs.
  3. siRNAs are loaded into the RISC (RNA-induced silencing complex).
  4. RISC uses one strand (the guide strand) to find complementary mRNAs.
  5. Perfect match (siRNA): mRNA is cleaved and degraded.
  6. Partial match (miRNA): Translation is repressed or the mRNA is destabilized.
• Differences between siRNA and miRNA outcomes:
  • siRNA:
    • Often exogenous.
    • Targets mRNA with perfect complementarity → cleavage and degradation.
  • miRNA:
    • Endogenous.
    • Targets mRNA with partial complementarity → repression of translation or destabilization.
• Where does miRNA come from?
  • Transcribed as primary miRNA (pri-miRNA) → processed in nucleus by Drosha → pre-miRNA → exported to cytoplasm → processed by Dicer → mature miRNA.
• RdRP (RNA-dependent RNA polymerase):
  • Amplifies RNAi signals by synthesizing more dsRNA from target mRNA.
  • Enhances gene silencing by generating additional siRNAs.
• How can dsRNA be introduced?
  • Injected directly into the cell.
  • Delivered via viral vectors.
  • Expressed from transgenes designed to form hairpin RNAs.

CRISPR-Cas Systems

• CRISPR Locus Components:
  • Repeats: Identical sequences.
  • Spacers: Unique sequences derived from phage DNA (past infections).
  • cas genes: Encode the enzymes (Cas proteins) involved in the process.
• Protospacers:
  • DNA sequences in viruses that match CRISPR spacers.
  • When acquired by CRISPR, they become new spacers in the CRISPR array.
• crRNAs (CRISPR RNAs):
  • Transcribed and processed from the CRISPR array.
  • Guide Cas proteins to target DNA by base-pairing with protospacer sequences.
• Cas proteins:
  • Nucleases that cut DNA (e.g., Cas9).
  • Use crRNA to locate target DNA.
• Class 1 vs Class 2 CRISPR Systems:
  • Class 1: Use multi-protein effector complexes (e.g., Cascade).
  • Class 2: Use single-protein effectors (e.g., Cas9, Cas12).
• tracrRNA and sgRNA:
  • tracrRNA: Binds pre-crRNA and helps in maturation and loading onto Cas9.
  • sgRNA (single-guide RNA): Lab-designed fusion of crRNA and tracrRNA; directs Cas9 to specific DNA for editing.
• Genome editing with Cas9:
  • Cas9 makes double-stranded break.
  • DNA repair by:
    • NHEJ (non-homologous end joining) → insertions/deletions (knockout).
    • HDR (homology-directed repair) → precise insertion (knock-in).
• dCas9:
  • Catalytically dead Cas9.
  • Can be fused to transcriptional activators or repressors.
  • Used to repress (CRISPRi) or activate (CRISPRa) gene expression without cutting DNA.

Other Gene Editing Systems

• Zinc Finger Nucleases (ZFNs):
  • DNA-binding domains: zinc fingers (bind 3-bp DNA sequences).
  • Fused to FokI nuclease.
  • Dimerize to cut DNA.
  • Require careful design for specificity.
• TALENs:
  • Transcription Activator-Like Effectors (TALEs) bind specific DNA sequences.
  • Also fused to FokI.
  • Easier to design than ZFNs (one TALE = one base).
• Peptide Nucleic Acids (PNAs):
  • Synthetic molecules that mimic DNA/RNA.
  • Bind to complementary DNA/RNA.
  • Can block transcription or translation by interfering with base-pairing.

Analysis of Gene Expression

• Understanding gene expression means measuring when, where, and how much a gene is transcribed or translated. Focuses on reporter systems, regulatory region analysis, protein-DNA interaction assays, and high-throughput expression profiling.

1. Reporter Genes

A reporter gene is a gene whose product is easily measurable, and is used as a proxy to detect transcriptional or translational activity of a gene or regulatory element.

• Common Reporter Systems:
  • β-galactosidase (lacZ):
    • What is measured: Enzymatic activity.
    • What you observe: Blue color when X-gal is added (indicates expression).
  • Luciferase (luc):
    • What is measured: Light production.
    • What you observe: Bioluminescence – light detected with imaging systems.
  • Green Fluorescent Protein (GFP):
    • What is measured: Fluorescence.
    • What you observe: Green fluorescence under UV/blue light; visible under microscope.
  • β-lactamase:
    • What is measured: Antibiotic resistance.
    • What you observe: Growth in presence of ampicillin (if expressed).
• Reporter genes are often fused to regulatory sequences of interest to study when/where/how strongly a gene is expressed.

2. Gene Fusions and Regulatory Analysis

• Gene Fusions:
  • Link a reporter gene to either:
    • Promoter/Enhancer regions: To monitor transcriptional activity.
    • Coding sequences: To monitor protein localization or translation.
  • Used to dissect regulatory sequences that control gene expression.
• Deletion Analysis:
  • Involves making systematic deletions in the promoter/enhancer region to determine which sequences are required for transcription.
  • Example:
    • Deleting a region causes reporter activity to drop → that region contains an essential enhancer or TF binding site.

3. DNA-Protein Interaction Assays

• These assays help identify whether a specific protein binds DNA and where it binds.
• Gel Shift Assay (EMSA – Electrophoretic Mobility Shift Assay):
  • Labeled DNA fragment is incubated with protein.
  • If protein binds, the DNA-protein complex moves slower in gel (shifted band).
  • Can include:
    • Unlabeled competitor DNA (to confirm specificity).
    • Antibody to create a "super shift", confirming protein identity.
• Footprinting Analysis:
  • DNA is labeled on one end and incubated with or without protein.
  • Digested with DNase I, which cuts unprotected DNA.
  • Protein protects its binding site → no cuts where protein binds = footprint.
  • Tells you exact location of protein binding on DNA.

4. Chromatin Immunoprecipitation (ChIP)

• Standard ChIP:
  • Crosslink protein-DNA complexes in living cells.
  • Fragment chromatin (e.g., sonication).
  • Use antibody against the protein of interest to pull down DNA-protein complexes.
  • Reverse crosslinks and purify DNA.
  • Analyze by PCR, qPCR, or sequencing.
  • ChIP tells you what DNA sequences are bound by a specific protein in vivo.
• ChIA-PET (Chromatin Interaction Analysis by Paired-End Tagging):
  • Combines ChIP with high-throughput sequencing.
  • Captures 3D interactions between DNA regions mediated by protein complexes.
  • Used to identify enhancer-promoter loops and long-range regulation.

5. Transcription Start Site Mapping

• These techniques help determine where transcription begins on a gene.
• Primer Extension:
  • Use a labeled primer that anneals to RNA.
  • Reverse transcriptase extends primer to 5' end of RNA.
  • Run product on a gel → length tells you the start site.
• S1 Nuclease Mapping:
  • Hybridize RNA to a labeled ssDNA probe.
  • S1 nuclease digests single-stranded DNA.
  • Protected duplex region = exact start point of RNA.

6. High-Throughput Gene Expression Profiling

• RNA-Seq:
  • Total or polyA+ RNA is converted to cDNA.
  • Sequenced using next-gen sequencing (e.g., Illumina).
  • Reads are mapped to genome to:
    • Quantify gene expression.
    • Detect alternative splicing, new transcripts, noncoding RNAs.
  • RNA-Seq gives global, quantitative, and highly sensitive expression data.
• DNA Microarrays:
  • Thousands of DNA probes are fixed on a chip.
  • Sample RNA → convert to labeled cDNA → hybridize to chip.
  • Fluorescent signal indicates presence and abundance of mRNA for each gene.
• Microarray Fabrication:
  • On-chip synthesis:
    • DNA sequences are built directly on the chip.
    • Uses light-sensitive masks or virtual masks (digital patterns).
  • Printed arrays:
    • Pre-made DNA spots printed on the slide.
  • Microarrays compare expression across conditions (e.g., normal vs cancer cells).

7. Quantitative PCR (qPCR) – TaqMan Assay

• Detects and quantifies specific mRNA levels in real time.
• Uses:
  • Reverse transcription to convert RNA → cDNA.
  • TaqMan probe:
    • Has fluorescent reporter and quencher.
    • During PCR, probe is cleaved by Taq polymerase → fluorescence increases.
• Output: Ct (threshold cycle) – lower Ct = more RNA.
• Very sensitive and specific for targeted gene expression analysis.

Summary Chart

Plasmids

• Plasmids are double-stranded, circular DNA molecules found in bacteria and sometimes in eukaryotes.
• They self-replicate within host cells, separate from the host chromosome.
• Most plasmids are circular, but some rare ones are linear with protected ends (e.g., proteins or hairpin structures).

Plasmid Replication Mechanisms

1. Bidirectional Replication
   • Similar to chromosomal replication.
   • Two replication forks move outward from the origin of replication (ori) in opposite directions.
2. Rolling Circle Replication
   • Nick made on one DNA strand at the ori.
   • DNA polymerase extends the 3’ end, displacing the old strand as it synthesizes a new one.
   • The displaced single strand is then used as a template to make a new complementary strand.
   • Result: 2 double-stranded plasmids.

Plasmid Incompatibility

• Two plasmids are incompatible if they share the same origin of replication or replication machinery.
• Incompatible plasmids cannot stably coexist in the same cell — one will eventually be lost.
• Compatible plasmids have different origins and can be maintained together.

Plasmid Copy Number Regulation – RNAII and RNAI

• RNAII: Produced from the plasmid; serves as a primer for DNA replication.
• RNAI: Antisense RNA that binds RNAII and prevents it from forming the primer.
• When RNAI binds RNAII:
  • Forms a duplex → RNase H cannot cleave RNAII → No primer → No replication.
• More RNAI = fewer plasmid copies.
• This regulatory mechanism influences plasmid compatibility as well (shared control → incompatibility).

Plasmid-Encoded Antibiotic Resistance

Selfish Plasmids – Toxin/Antidote Systems

• Some plasmids ensure retention by killing host cells that lose them.
• Encode:
  • Toxin (stable protein).
  • Antidote (unstable protein or RNA).
• If the plasmid is lost:
  • Antidote degrades quickly.
  • Toxin remains and kills the host.

Toxin-Encoding Plasmids

• ColE1 and ColE2 Plasmids
  • Encode bacteriocins (colicins) – toxic proteins targeting other bacteria.
  • Colicin E1: Forms pores in target cell membranes → disrupts ion balance.
  • Colicin E2: A DNase that cleaves target bacterial DNA.
  • Both plasmids carry immunity genes that protect the host cell from its own toxin.
• Ti Plasmid – Tumor Inducing in Plants
  • Found in Agrobacterium tumefaciens.
  • Transfers a T-DNA region into plant cells.
  • T-DNA genes include:
    • Auxin and cytokinin synthesis → uncontrolled cell division → tumor (gall).
    • Opine synthesis → nutrients the bacteria can use.

Viruses

• A virus is a non-living infectious particle composed of:
  • Genome: DNA or RNA.
  • Capsid: Protein coat that surrounds genome.
  • Envelope (sometimes): Lipid membrane derived from host.
• A virion is a complete, infectious viral particle.

Viral Lifecycle

1. Attachment: Virus binds to host surface receptors.
2. Entry:
   • Bacteriophages inject genome.
   • Enveloped viruses fuse membranes and enter whole.
3. Replication:
   • Genome is copied using host machinery.
4. Transcription & Translation:
   • Early genes: replication proteins.
   • Late genes: structural proteins.
5. Assembly: Capsid proteins and genome packaged.
6. Release: Often via lysis or budding.

Hershey-Chase Experiment

• Used radioactive labeling:
  • $^{35}S$ labeled protein (capsid).
  • $^{32}P$ labeled DNA.
• Found that only DNA entered the host cell during infection.
• Showed that DNA is the genetic material.

Viral Growth Patterns

• Lytic: Virus replicates, kills host, releases virions.
• Lysogenic/Latent: Viral genome integrates into host DNA, replicates with it.
  • Can switch to lytic under stress.

Virus Diversity

• Genome types:
  • DNA or RNA.
  • Single- or double-stranded.
  • Linear or circular.
• Structures:
  • Spherical, filamentous, complex (e.g., bacteriophages).
  • With or without envelope.
• Overlapping Genes
  • Common in small viral genomes.
  • Multiple genes use different reading frames or share DNA.
  • Evolve together: mutations affect multiple proteins.
• Mutation Rate
  • RNA viruses have higher mutation rates.
  • Lack of proofreading by RNA-dependent RNA polymerase.

RNA Viruses: Plus vs Minus Strand

Retroviruses (e.g., HIV)

• Genome in virion: 2 single-stranded RNAs.
• Uses reverse transcriptase to convert RNA → DNA.
• Integrates into host genome.
• Remains in the host genome for life.
• Key genes:
  • gag: structural proteins.
  • pol: reverse transcriptase, integrase.
  • env: envelope proteins.
• tRNAs: Bind to RNA genome, serve as primers for reverse transcription.

Transposons – Mobile Genetic Elements

• Found within larger DNA molecules (e.g., chromosomes, plasmids).
• Do not self-replicate.
• Move using transposase enzyme.
• Cause mutations and genome rearrangements.

Structure of a Transposon

• Inverted repeats at ends.
• Transposase gene: Encodes enzyme for movement.
• Some complex ones also carry antibiotic resistance or other genes.

Two Types of Transposition

• Resolvase and internal resolution sites (IRS) help separate joined DNAs in replicative transposition.

Composite Transposons

• Formed when two simple transposons bracket additional genes.
• Move as one unit.
• Over time, redundant sequences may be lost → becomes a single composite transposon.

Regulation of Transposition

• Transcriptional regulation via upstream regulator (orfA).
• Translational regulation: transposase gene requires a frameshift → rare event → limits activity.

Genome Rearrangement by Transposons

• Can invert, delete, or move host DNA when jumping.
• Can disrupt genes or regulatory regions → affect gene expression.

Ac and Ds Elements (Corn)

• Ac (Activator):
  • Has functional transposase → can move itself and Ds elements.
• Ds (Dissociation):
  • Mutated/lack transposase → cannot move on their own.
  • Can move only if Ac is present in the same cell.

Retrotransposons

• Move via RNA intermediate.
• Use reverse transcriptase, similar to retroviruses.
• Examples:
  • Ty1 in yeast.
  • LINEs (Long Interspersed Elements): Encode reverse transcriptase.
  • SINEs (Short Interspersed Elements): Non-autonomous, often derived from other RNAs (e.g., Alu from 7SL RNA).
  • Pseudogenes: Processed mRNAs reverse transcribed and inserted into genome; lack introns and promoters → nonfunctional.