Lecture 2 (Erhardt) Flashcards
Transcription
RNA Polymerase Structure & different σ-factors
RNA Polymerase Structure
- Core-enzyme: consisting of a, ß, ß’, omega Subunits
- Holo-enzyme: σ-factor
σ⁷⁰: Housekeeping factor. It controls the transcription of essential genes
σ³²: Activated in response to heat shock or other stress conditions like high temperatures, which can cause protein misfolding
σ²⁸: Controls the expression of genes involved in flagella synthesis and motility
σ³⁸: Active during the stationary phase of bacterial growth or under nutrient-limited conditions
σ⁵⁴: Regulates genes involved in nitrogen assimilation when nitrogen sources are limited
Bacterial Promotor Structure
Bacterial Promotor Structure
- Upstream of the transcription start
- RNA Polymerases binds here
- -35 region: TTGACA
- -10 region: Pribnow Box (TATAAT)
Bacterial Operon Structure
Bacterial Operon Structure
- Functioning unit of DNA containing multiple genes under the control of a single promoter
Contains:
- Promotor: DNA sequence upstream of the structural genes where RNA polymerase binds to initiate transcription
- Operator: short DNA region located between the promoter and the structural genes. It acts as a regulatory switch, controlling whether transcription happens. A repressor protein can bind to the operator to block RNA polymerase, preventing transcription
- Structural Genes: code for proteins
- Regulatory Gene: codes for a repressor or activator protein that influences the operon’s activity. It is usually located outside of the operon
Transcription
Initiation
Transcription Initiation
- RNA polymerase holoenzyme scans DNA for promotor sequences
- Sigma-factor in the RNA polymerase recognizes and binds to the promotor
- RNA transcription begins at nucleotide +1
- RNA polymerase unwinds DNA and begins transcribing RNA with rNTPs (ribonucleoside triphosphates)
- Sigma-factor leaves the complex
Regulation of Transcription
1: Anti-sigma factor proteins
Anti-sigma factor proteins
- Some sigma factors are controlled by antisigma factor proteins that inhibit activity by binding directly to specific sigma factors and blocking their access to the RNA polymerase.
- The release of sigma factors from their anti-sigma factor proteins is important for regulating spore formation and flagella construction
- Anti-sigma factors can themselves be neutralized by anti-anti-sigma factors
Example of Anti-sigma factors
Regulation of flagellar gene expression
Regulation of flagellar gene expression
-
FliA (σ²⁸) Inactive Due to FlgM:
FliA is initially inactive because it is bound by the anti-sigma factor FlgM, preventing it from initiating transcription. -
Basal Body and Hook Formation:
The bacteria construct the flagellum’s hook and basal body. During this phase, FlgM keeps FliA inactive, delaying the synthesis of late-stage flagellar genes. -
FlgM Export:
After the basal components are complete, FlgM is exported out of the cell, freeing FliA to bind with the core RNA polymerase. -
FliA Activates Late Genes:
FliA then activates transcription of late-stage genes, including those for flagellin, motor proteins, and chemotaxis machinery.
Regulation of Transcription
2: Trancription Factors
Transcription Factors
- The initiation of transcription is controlled by
regulatory proteins = transcription factors:
- Bind DNA at or near gene promoters and stimulate or prevent binding of RNA polymerase to promoter
- DNA-binding domain interacts with major groove of DNA and often form dimers
Regulation of Transcription
2A: Repressors
Repressor Protein: A molecule that binds to the operator to prevent gene expression
Scenario 1: Induction by Repressor Release
- A repressor protein binds to DNA and blocks transcription, keeping the gene inactive.
- When a Ligand (inducer) binds to the repressor, it changes shape and releases the DNA.
- This allows transcription to resume, leading to gene expression
Scenario 2: Derepression by Repressor-Corepressor Complex
- A repressor only binds to DNA when a corepressor molecule attaches to it, forming a complex that blocks transcription
- When the corepressor is removed, the complex dissociates from the DNA.
- This allows transcription to restart, enabling gene expression.
Regulation of Transcription
2B: Activators
Activator Protein: enhances gene expression of specific genes
Ligand (Inducer): activates the activator protein, enabling it to bind DNA and start transcription.
How does ChIP-Seq work and what is it used for?
Chromatin Immunoprecipitation Sequencing
ChIP-Seq: combines chromatin immunoprecipitation (ChIP) with sequencing technology to identify DNA-protein interactions across the genome
Procedure:
- Crosslinking: Cells are treated with chemicals to crosslink proteins to DNA
- Shearing: The DNA is broken into small fragments
- Immunoprecipitation: An antibody specific to the protein of interest with beads (e.g., a transcription factor) is used to pull down the DNA-protein complex
- Purification: DNA bound to the protein is purified from the other fragments through the beads and the crosslinking is reversed
- Sequencing: The purified DNA fragments are sequenced to identify the DNA regions bound by the protein
- Applications:
- Identifying binding sites of transcription factors
- Mapping histone modifications
- Understanding gene regulation and chromatin structure
Key Components of the lac operon
Example for Regulation of Gene Expression
Structural Genes:
lac Z: Encodes β-galactosidase, an enzyme that breaks down lactose into glucose and galactose.
lac Y: Encodes lactose permease, a protein that transports lactose into the cell.
lac A: Encodes thiogalactoside transacetylase
Regulatory Elements:
Promoter: A DNA sequence where RNA polymerase binds to start transcription of the lac operon.
Operator: A DNA segment that acts as a binding site for the lac repressor protein, controlling access to the operon.
lacI Gene: Located upstream of the lac operon with it’s own promotor, it encodes the lac repressor protein, which regulates the operon.
Lac Operon Regulation
Example for Regulation of Gene Expression
Lac Operon Regulation
No Lactose Present:
- The lac repressor (produced by lacI) binds to the operator.
- This blocks RNA polymerase from transcribing the operon, preventing unnecessary expression.
Lactose Present
- Lactose is converted to allolactose (an inducer), which binds to the lac repressor, causing it to release the operator.
- RNA Polymerase can bind to the operator and express the lacZ,Y,A genes
- Low glucose levels lead to an increase in cAMP concentration, which binds with CAP (Catabolite Activator Protein) to form the CAP-cAMP complex.
- The CAP-cAMP complex binds near the promoter, enhancing RNA polymerase binding by interacting with the αCTD and increasing transcription of the operon.
Lactose Present, Glucose Present:
- High glucose levels result in inhibition of the enzyme adenylate cyclase, which is responsible for converting ATP to cAMP, which in result lowers cAMP concentrations
- Without cAMP, the CAP-cAMP complex cannot form, and CAP does not bind near the promoter.
- This reduces transcription, as RNA polymerase binding is not as strongly facilitated, even if the repressor is inactive
Diauxic Growth Phenomenon
Key Concepts in Diauxic Growth:
- Catabolite Repression: When glucose is present, the lac operon is repressed, even if lactose is available. This mechanism ensures glucose is used first.
- Induction of the Lac Operon: When glucose is depleted, CAP-cAMP binds to the promoter region, activating the lac operon in the presence of lactose.
- Two-Phase Growth Curve: The growth curve shows a rapid growth phase on glucose, a lag phase while the bacteria switch to lactose metabolism, and a slower growth phase on lactose.
Regulation of Transcription
3: Riboswitches
Riboswitches
- Allow bacteria to control gene expression in response to small molecules directly
Left Side (No Ligand Bound):
- In the absence of the ligand, the riboswitch forms an antiterminator structure, allowing RNA polymerase to continue transcription through the coding sequence.
Right Side (Ligand Binding):
- When a specific ligand binds to the aptamer region of the riboswitch, it causes the mRNA to fold differently, forming a terminator structure.
- This terminator structure includes a hairpin loop followed by a series of uracils (U’s), which acts as a transcription terminator.
- The terminator structure causes transcription to stop prematurely, preventing the expression of the downstream gene.
Regulation of Transcription
Transcription Termination
Rho-dependent termination
Rho-Dependent Termination
Binding of Rho:
- The Rho protein binds to the rut site (c-rich region) on the RNA and starts moving along the RNA strand toward the RNA polymerase at the 3’ end of the transcript.
RNA Threading and Movement:
- Rho moves along the RNA, following behind the RNA polymerase, which pauses at a pause site downstream from the rut site.
- Rho “pulls” itself toward the RNA polymerase by threading the RNA through its hexamer structure.
Termination:
- When Rho catches up with RNA polymerase, the contact between Rho and RNA polymerase causes RNA polymerase to release the RNA transcript and dissociate from the DNA, ending transcription.
Regulation of Transcription
Transcription Termination
Rho-Independent (Intrinsic) Termination
Rho-Independent (Intrinsic) Termination
- As RNA polymerase transcribes a GC-rich region in the DNA, the RNA forms a stable hairpin loop structure due to complementary base pairing within the GC-rich sequence.
- NusA protein binds to RNA polymerase and interacts with the hairpin structure, helping to stabilize this hairpin loop.
Poly-U Tail:
- Following the GC-rich region, there is a stretch of adenine (A) nucleotides in the DNA, which results in a sequence of uracils (U’s) in the RNA.
- This U-rich sequence forms a weak interaction with the DNA (A-U base pairs are weaker than G-C pairs).
Termination:
- The hairpin loop causes RNA polymerase to pause.
- The weak A-U bonds in the RNA-DNA hybrid make it easier for the RNA transcript to detach from the DNA.
- The combined effect of the hairpin loop and the weak A-U bonds leads to the release of the RNA and termination of transcription.
Antitermination
Antitermination
- A regulatory process that allows RNA polymerase to bypass normal termination signals in DNA, resulting in the production of extended RNA transcripts.
Mechanism:
- Antiterminators (specialized proteins) interact with RNA polymerase or the newly synthesized RNA to modify its behavior.
- These modifications prevent the RNA polymerase from pausing or detaching at typical termination sites, allowing it to continue transcribing downstream genes.
- This process may involve the recognition of specific sequences in the RNA or the recruitment of additional protein factors that stabilize RNA polymerase on the DNA template
Functions and Importance:
- Antitermination allows for the controlled expression of genes that are clustered together, often as operons, by enabling the transcription of multiple genes in a sequence.
- It plays a role in bacterial responses to environmental changes, ensuring that essential genes can be expressed under specific conditions
Antitermination
Example: Attenuation of the trp operon
trp Operon
- has a leader sequence (trpL) that forms secondary structures in the mRNA, depending on the levels of tryptophan in the cell.
- This leader sequence contains four regions that can form different stem-loop structures, including a terminator (transcription stops) or antiterminator (transcription continues)
High Tryptophan Levels
- Ribosomes translates through trp codon of leader mRNA and encounters translation stop codon
- Ribosome stops, covering mRNA regions 1 and 2, RNA Polymerase continues to transcribe regions 3 and 4
- Formation of a terminator loop between region 3 and 4 in the mRNA
- This loop binds RNA polymerase and causes its release, preventing expression of the downstream genes (trpE) needed for tryptophan synthesis
Low Tryptophan Levels
- Ribosomes stall at trp codons during translation of the leader peptide because of a lack of tRNA tryptophan.
- This allows for the formation of an antiterminator loop between region 2 and 3, preventing the terminator structure from forming.
- RNA polymerase can then continue transcribing the downstream genes, allowing the cell to produce tryptophan
Shine Dalgarno Sequence
Shine Dalgarno Sequence
- short, conserved sequence of nucleotides in mRNA, located 5-10 nucleotides upstream of the start codon (AUG).
- serves as a ribosome binding site and helps align the ribosome with the start codon for translation initiation.
- Typically rich in purines (e.g., 5’-AGGAGGU-3’ for E.coli).
- Function: Base-pairs with the 16S rRNA in the small ribosomal subunit, ensuring proper positioning of the mRNA for translation.
What does up- and downstream refer to?
Upstream:
- Refers to the direction toward the 5’ end of the DNA strand.
- Region of DNA before the gene or transcription start site
- Promoters are typically found upstream of the genes they regulate
Downstream:
- Refers to the direction toward the 3’ end of the DNA strand
- Region of DNA after the gene or transcription start site
- The RNA transcript, which is synthesized during transcription, extends downstream from the transcription start site
Classes of RNA in Bacteria
mRNA: Carries genetic information from DNA to the ribosome for protein synthesis.
rRNA: Forms the structural and catalytic components of ribosomes.
tRNA: Delivers specific amino acids to the ribosome during translation.
sRNA: Regulates gene expression at the RNA level.
tmRNA (Hybrid between tRNA and messanger RNA): Rescues stalled ribosomes and tags incomplete proteins for degradation.
Ribozymes: RNA molecules that catalyze specific chemical reactions.
crRNA: Guides bacterial immune response against foreign DNA.
