Lecture 3 (Erhardt) Flashcards
Regulation of protein synthesis & Molecular and cell biology techniques
Regulation of mRNA stability and Translation
1A: Small regulatory RNAs (sRNAs)
Small regulatory RNAs (sRNAs)
- Short (100–200 nt) non-coding RNAs encoded by intergenic sequences.
- Regulate mRNAs by base pairing with target sequences, either promoting or inhibiting translation.
- Can block ribosome binding near the RBS (with the help of Hfq protein) or recruit RNases to degrade the mRNA.
- May also stabilize mRNAs by protecting them from degradation, enhancing translation.
Functions
- Stress Response: sRNAs regulate mRNAs involved in stress adaptation, allowing quick responses to environmental changes.
- Nutrient Availability: sRNAs control genes for nutrient uptake and metabolism, optimizing resource use based on availability.
Identification of sRNAs (small RNAs)
sRNA identification:
1) Cell Preparation: Bacterial cells are grown to an early stationary phase for optimal sRNA expression
2) RNA Isolation and Enrichment:
- Small RNA Enrichment (<500 nt) followed by RNA-Seq identifies sRNAs
- Primary Transcript Enrichment (TEX) focuses on unprocessed RNAs for genome-wide transcription start site (TSS) identification
3) Chromatin Analysis (ChIP-chip): ChIP-chip maps RNA polymerase and regulatory factors, linking sRNA expression to chromatin context.
4) Data Integration: Combines RNA-Seq and ChIP data to identify sRNAs, their promoters, and transcribed regions.
Regulation of mRNA stability and Translation
1B: Cis-antisense RNAs (asRNAs)
Cis-antisense RNAs (asRNAs)
- non-coding RNA transcribed from the opposite DNA strand of a target gene, resulting in a complementary sequence to the target mRNA.
- Base Pair with their complementary target mRNA and form RNA-RNA duplexes
- Binding of an asRNA to its target mRNA can prevent ribosome access and block translation
- Duplex formation may make the mRNA more susceptible to degradation by RNases, reducing gene expression.
- In some cases, asRNAs can protect their target mRNA from degradation, increasing stability and enhancing expression
Cis-antisense RNAs (asRNAs)
Example
S-box Riboswitch Role
- The S-box riboswitch controls antisense RNA (asRNA) transcription based on SAM levels.
- This regulation ensures that cysteine synthesis is tied to SAM availability.
Low SAM Levels
- SAM does not bind to the S-box riboswitch.
- RNA polymerase continues transcription, producing antisense RNA (asRNA).
- Collisions between RNA polymerases can cause premature termination and degradation of incomplete RNA transcripts.
- This disruption can interfere with normal gene expression.
High SAM Levels
- SAM binds to the S-box riboswitch.
- The riboswitch forms a transcription termination loop, halting antisense RNA production.
- RNA polymerase completes the synthesis of ubiG-mccB-mccA mRNA.
- Normal gene expression proceeds, allowing for the production of cysteine.
Regulation of Translation
2: Translation Initiation by Riboswitches
Translation Initiation by Riboswitches
- mRNAs form secondary structure and with help of a ligand (small molecule or second messenger) this structure can be changed
- Covers the translation start (SD-sequence) and prevents ribosome from binding
- Riboswitches can function as RNA thermometers
- With temperature increase this structure loses binding and start sequence gets free
Regulation of Translation
3: Rescue of stalled Ribosomes by tmRNA (transfer-messenger RNAs)
Rescue of stalled Ribosomes
- If the mRNA is partially degraded (stop codon missing), the Ribosome can get trapped
- Rescue molecule tmRNA gets into the A-site of the ribosome (where a normal tRNA would enter), which then hops on the mRNA part of the tmRNA and continue translation there
- the tmRNA carries a short sequence encoding a peptide tag (SsrA tag) that marks the protein for degradation
- The ribosome resumes translation, using the tmRNA template, and synthesizes this tag onto the end of the partially completed protein
- The tagged protein gets recognized and unfolded by CIpX and ClpP
- Energy landscape of protein folding –> local energy minima –> can get stuck –> Molecular chaperones software to predict folding alpha-fold
Protein Unfolding by ClpXP
ClpXP protease complex
Substrate Engagement
- The ClpX subunit binds and recognizes the ssrA-tagged protein
- ATP is hydrolyzed to power the engagement and initial binding to the substrate.
Mechanical Unfolding
- ClpX uses energy from further ATP hydrolysis to mechanically unfold the tagged protein
- The unfolded protein is progressively pulled through the ClpX pore
Translocation and Degradation
- The unfolded protein is translocated into the ClpP protease chamber
- Inside ClpP, the polypeptide is degraded into small peptides
Regulation of Translation
4: Molecular Chaperones
Molecular Chaperones
- Are a group of proteins that assist in the proper folding, assembly, maintenance, and the refolding or degradation of proteins
- Help newly synthesized or partially unfolded proteins attain their correct structure
- Bind to exposed hydrophobic regions of nascent or misfolded proteins to prevent them from clumping together and forming aggregates, which can be toxic to the cell.
- Can refold misfolded proteins that arise due to cellular stress or mutations
- If a protein cannot be correctly folded, chaperones can target it for degradation by the cell’s protein degradation machinery (e.g., proteasomes)
Molecular Chaperones
Example Trigger Factor, DnaK/DnaJ, and ClpB
Molecular Chaperones
1) Trigger Factor binds to newly synthesized proteins as they emerge from the ribosome, forming a protective cavity to support initial folding and prevent aggregation.
2) After initial folding, some proteins may misfold or form aggregates. DnaJ delivers misfolded proteins to DnaK, which binds and helps refold them through ATP-driven cycles.
3) If aggregates form, ClpB works with the DnaK system to break apart and refold or degrade the protein clusters, preventing harmful accumulation
Molecular and Cell biology techniques
1: Optogenetics
Optogenetics
- A technique that enables precise control of cellular processes using light
a. Native AraC Regulation
- AraC tf controls the P_BAD promoter in the presence of arabinose
- Without arabinose, AraC binds to operator sites O1 and O2, forming a DNA loop that blocks RNA polymerase.
- Arabinose binding disrupts this loop, allowing RNA polymerase to initiate transcription.
b. Light-Inducible AraC
- Native AraC protein contains an arabinose-binding and DNA-binding domain.
- By engineering AraC with a light-inducible domain, the protein’s activity can be regulated by light instead of arabinose
c. Light-Controlled Transcription
- In darkness, AraC represses transcription.
- Upon light exposure, AraC changes conformation, permitting RNA polymerase to bind and activate transcription at P_BAD.
Molecular and Cell Biology techniques
2: Recombineering
Recombineering
- A technique for precise DNA modifications using homologous recombination
Overview of the Technique
1) Preparation of Linear DNA
- Exo (Exonuclease) binds to a linear DNA fragment, such as a PCR product or ds DNA
- Exo degrades the 5’ ends of the DNA, creating 3’ single-stranded overhangs
2) Binding of Beta Protein
- The Beta protein binds to the exposed 3’ ends and helps stabilize them, while promoting base pairing with complementary regions in the target DNA
3) Homologous Recombination
- The single-stranded DNA regions of the linear DNA fragment align with homologous sequences in the target DNA
- Homologous recombination occurs, resulting in the incorporation of the desired DNA changes into the target DNA
Molecular and Cell Biology techniques
2: Recombineering: Reporter Gene Fusion Example
Transcriptional (Operon) Fusion
- In a transcriptional fusion, the gfp gene is placed downstream of a target gene’s promoter and has it’s own ribosome-binding site but is controlled by the target genes promotor
- This allows transcription from the target gene’s promoter, producing one transcript but resulting in separate proteins.
Translational (Gene) Fusion
- In a translational fusion, the gfp gene is fused directly to the coding sequence of the target gene without its own ribosome-binding site, resulting in a single, fused hybrid protein.
Molecular and Cell Biology Techniques
3A: Transposon Mutagenesis
Transposon Mutagenesis
- Uses transposons (mobile genetic elements flanked with inverted repeats) to induce mutations in a genome.
- This allows for the disruption and study of gene function
Process Overview
- Insertion: A transposon carrying a selectable marker (e.g., antibiotic resistance) is introduced into the bacterial genome or target DNA
- Selection: Cells with successful transposon insertions are selected based on their resistance to antibiotics or another selectable trait
- Characterization: The insertion site is mapped and studied to determine how the disruption affects gene function. This can reveal gene essentiality, pathways, and functional relationships
Molecular and Cell Biology Techniques
3B: Transposon Mutagenesis: Transposon Insertion Sequencing
Transposon Insertion Sequencing
Generating and Testing Transposon Libraries
- Library Creation: A high-density library of mutants with random transposon insertions is generated, disrupting genes throughout the genome.
- Condition Testing: The mutant pool is grown under various conditions. This step reveals which mutations affect growth and survival
- Sequencing and Analysis: DNA from the library is extracted, and high-throughput sequencing is performed to identify and count transposon insertion sites, providing insights into which genes are essential or non-essential under each condition
Data Analysis and Gene Identification
- Read Counts and Mapping: For each condition, the frequency of transposon insertions is mapped. Genes with reduced or no insertions under a specific condition are likely essential for growth in that environment.
- Condition Comparison: By comparing insertion patterns between different conditions, genes important for specific environmental adaptations or processes can be identified
- Saturation Analysis:
High Saturation: Indicates comprehensive coverage of the genome, providing robust data on essential and non-essential genes.
Low Saturation: Suggests fewer disrupted sites per gene, offering less comprehensive but still useful information.
Molecular and Cell Biology Techniques
4A: Transcriptome analyses: NGS
Next-Generation Sequencing (NGS)
-
Fragmentation & Adapter Ligation
DNA is cut into fragments, and special adapters are attached to the ends. -
PCR Amplification
The amount of DNA fragments is increased using PCR. -
Binding to Flow Cell
DNA is sequenced on a glass surface (flow cell) where oligonucleotides that match adapter sequences are bound. - Library Denaturation & Binding DNA fragments in the library are denatured to form single-stranded DNA and added to the flow cell. The forward strand binds to one of the oligonucleotides, and the reverse strand is synthesized. The forward strand is washed away, leaving the library bound to the flow cell.
-
Cluster Amplification
Clonal amplification occurs through PCR, forming clusters of identical DNA strands. -
Bridge Amplification
Single strands bind to a second oligonucleotide on the flow cell, creating a bridge structure, which is then copied. The strands are denatured to form clusters, and reverse strands are cut and washed away, leaving only forward strands. -
Sequencing
A sequencing primer binds to the forward strand. Fluorescently tagged nucleotides and terminators are added. One base is incorporated at a time, and a camera captures the color emitted by each cluster. Terminators are removed, allowing the next nucleotide to be added, and the process repeats until the sequence is completed. -
Read Demultiplexing & Mapping
Reads are sorted based on attached indexes and mapped to a reference genome for analysis.
Molecular and Cell Biology Techniques
4B: Transcriptome Analysis: Dual RNA-seq
Dual RNA-seq of host and pathogen
Aim
- Gives information on which RNA is expressed when host is infected by pathogen
- By capturing transcriptomic changes in both organisms, dual RNA-seq can reveal how pathogens manipulate host cellular pathways and how the host mounts its defense.
Workflow
- Sample Collection: RNA is extracted from infected host tissues or cells.
- Library Preparation: RNA libraries for sequencing are prepared, often including steps to enrich for mRNA or deplete abundant host rRNA.
- Sequencing: High-throughput sequencing is performed to capture transcript data from both host and pathogen.
- Data Analysis: Bioinformatic tools differentiate and map reads to the respective host and pathogen genomes, followed by differential expression analysis.
Molecular and Cell Biology Techniques
4C: Transcriptome Analysis: Spatial transcriptomics of bacteria
2-step mRNA Fluorescence In Situ Hybridization (FISH)
Aim
- Detecting and visualizing specific mRNA molecules within fixed cells or tissue samples
Steps
Probe Hybridization
- involves using labeled RNA or DNA probes that are complementary to the target mRNA sequence.
- In the 2-step version, initial binding is achieved with an unlabeled primary probe that is specific to the target mRNA. This probe serves as a scaffold for subsequent detection.
Signal Amplification
- In the second step, labeled secondary probes bind to the primary probes, amplifying the signal and enhancing detection sensitivity.
- This step improves signal-to-noise ratio, allowing for more precise visualization of the mRNA molecules within cells.
- Labeling is often achieved using fluorescent dyes, enabling visualization under a fluorescence microscope.
