Exam 2 Flashcards
Describe basic gene structure
Promoter: Upstream regulatory region that binds transcription initiation complex.
5’ Untranslated Region (5’ UTR): Precedes the coding sequence, may contain regulatory elements.
Coding Sequence (Exons): Sequences that are transcribed into mRNA and translated into a protein.
Introns: Non-coding sequences removed from the primary transcript during RNA splicing.
3’ Untranslated Region (3’ UTR): Follows the coding sequence, may contain regulatory elements.
Terminator: Downstream sequence that signals transcription termination and release of mRNA.
coding sequence (continuous vs. non-continuous)
coding sequence contains DNA sequence used to produce the specific
protein encoded by the gene (will contain codons)
regulatory elements (promoter, terminator, enhancer, silencer)
regulatory elements are sequences of DNA that control transcription of a gene.
Usually proteins bind to these sequences to modulate transcription
Regulatory Elements:
Promoter: Upstream region that binds transcription initiation complex, initiates transcription.
Terminator: Downstream sequence that signals the end of transcription and releases RNA.
Enhancer: Can be upstream, downstream, or within introns. Binds transcription factors to increase transcription rate.
Silencer: Can be upstream, downstream, or within introns. Binds repressor proteins to inhibit gene expression
Describe transcription factor (general, activator, repressor)
Transcription factors are proteins that regulate transcription of a gene by
binding regulatory elements in a sequence specific manner
Transcription Factors:
General:
Proteins that bind to regulatory DNA sequences
Control transcription initiation, elongation, or termination
Activator:
Binds enhancers or promoters
Recruits transcription machinery
Increases transcription rate
Repressor:
Binds silencers or promoters
Blocks transcription machinery binding
Decreases or prevents transcription
Describe transcription start site (+1)
The nucleotide position where RNA polymerase begins transcribing a gene into mRNA.
Denoted as +1 in the DNA sequence.
Located within the promoter region, typically around 25-30 base pairs downstream of the promoter.
Marks the start of the transcribed region.
Accurate identification is crucial for determining promoter elements and gene structure
Describe proximal, distal, upstream, downstream
Proximal: Close to the gene or regulatory element being described. A proximal promoter is located near the transcription start site.
Distal: Far away from the gene or regulatory element. A distal enhancer can be thousands of base pairs away from the gene it regulates.
Upstream: The DNA sequence located before (5’ to) the gene or regulatory element. Upstream regulatory regions like promoters are found preceding the gene.
Downstream: The DNA sequence located after (3’ to) the gene or regulatory element. Downstream sequences like terminators follow the gene coding region.
In summary:
Proximal = close to
Distal = far from
Upstream = before
Downstream = after
coding strand
Coding strand (sense/non-template strand)
Same sequence as mRNA (except T→U)
Template for mRNA synthesis during transcription
Contains protein-coding sequence
Codons read 5’ to 3’ direction
Specifies amino acid sequence for translation
Other strand is template (antisense/non-coding)
template strand
- Template strand (antisense/non-coding strand)
- Complementary to coding/sense strand
- Used as template by RNA polymerase during transcription
- mRNA sequence is complementary to template strand sequence
- Does not directly encode protein sequence
- Contains anti-codons complementary to mRNA codons
- Provides template for mRNA synthesis
5’
Refers to the 5’ carbon in the sugar of a nucleic acid
Denotes the start/upstream end of a polynucleotide chain
Has a free phosphate group at the 5’ end
Read/synthesized in 5’ to 3’ direction
mRNA has 5’ cap for stability and translation
3’
Refers to the 3’ carbon in the sugar of a nucleic acid
Denotes the end/downstream end of a polynucleotide chain
Has a free hydroxyl group at the 3’ end
DNA synthesis occurs in 5’ to 3’ direction
mRNA has 3’ poly-A tail for stability and export
sense and antisense
Here are concise bullet points about sense and antisense for your cheat sheet:
Sense (Coding Strand):
- Same sequence as mRNA (except T→U)
- Template for mRNA synthesis
- Contains protein-coding sequence
- Codons read 5’→3’ direction
- Specifies amino acid sequence
Antisense (Template Strand):
- Complementary to sense/coding strand
- Used as template by RNA polymerase
- mRNA sequence complementary to antisense
- Contains anti-codons complementary to mRNA
- Does not directly encode protein
In summary:
- Sense = Coding, template for mRNA
- Antisense = Non-coding, template strand
coding region (coding
sequence)
Coding Region/Sequence:
- Portion of a gene that contains codons
- Codons specify amino acid sequence of protein
- Transcribed into mRNA
- Begins with a START codon (AUG)
- Ends with a STOP codon (UAA, UAG, UGA)
- Introns removed by splicing in eukaryotes
- Exons joined to form mature mRNA
- Does not include regulatory regions (promoters, etc.)
- Read in 5’ to 3’ direction during translation
In summary:
- Codes for protein product
- Transcribed region between START and STOP codons
- Exons make up coding sequence after splicing
Describe what happens and how in each stage of bacteria transcription
o Initiation, Elongation, and Termination
Initiation:
RNA polymerase binds to the promoter region of a gene
Promoter provides a recognition site for RNA polymerase
Transcription factors may assist RNA polymerase binding
DNA strand separation occurs, forming an “open complex”
RNA polymerase begins synthesizing RNA using ribonucleoside triphosphates
The first few nucleotides bind and form the transcription initiation complex
Elongation:
RNA polymerase moves along the template DNA strand
Ribonucleotides are added one by one to the 3’ end of the growing RNA transcript
The RNA transcript is extended in the 5’ to 3’ direction
RNA polymerase unwinds the DNA helix to expose the template strand
Transcription factors may regulate this process
RNA transcript is released from the exit channel of RNA polymerase
Termination:
RNA polymerase recognizes a terminator sequence in the DNA
This triggers a conformational change that destabilizes the transcription complex
The newly synthesized RNA transcript is released from RNA polymerase
RNA polymerase dissociates from the DNA template
Rho protein may aid in termination at certain terminator sequences
Generic overview transcription
Initiation: RNAP binds to promoter
Elongation: RNAP polymerizes mRNA complementary to DNA template sequence
Termination: A hairpin in the mRNA causes RNAP to dissociate from DNA
Transcription elongation:
RNAP adds RNA complementary to DNA
template and catalyzes a phosphodiester bond in 5’ to 3’ direction
RNA polymerase (RNAP)
enzyme that
polymerizes (bonds together) RNA nucleotides
that are complementary to the template sequence
Transcription initiation:
1) Sigma factor part of holoenzyme will
bind to the promotor in a sequence specific manner
Consensus sequence is the
most commonly found
sequence of nucleotides or
amino acids (across organisms
or within a genome)
Bacteria consensus promoter
sequence at -10 and -35 shown.
gene A
5’
3’
promoter RE
2) RNAP unwinds DNA double-helix and
3) sigma factor dissociates
Transcription termination:
terminator sequence causes hairpin to
form, which causes RNAP to dissociate
transcription elongation:
RNAP adds RNA complementary to DNA
template and catalyzes a phosphodiester bond in 5’ to 3’ direction
Know the parts of bacteria RNAP (RNA polymerase): core enzyme and sigma factor and
their functions
Bacterial RNA polymerase (RNAP) consists of two main components: the core enzyme and the sigma factor. Here are the parts and their functions:
Core Enzyme:
- Consists of 5 subunits: 2 α subunits, 1 β subunit, 1 β’ subunit, and 1 ω subunit
- Catalyzes the synthesis of RNA using the DNA template
- Contains the active site for nucleotide polymerization
- Responsible for elongation of the RNA transcript
Sigma (σ) Factor:
- A dissociable subunit that associates with the core enzyme
- Primary sigma factor (e.g., σ70 in E. coli) is required for initiation of transcription
- Recognizes and binds to specific promoter sequences on DNA
- Enables core enzyme to locate and bind to promoters
- Helps melt/unwind DNA for transcription initiation
- Dissociates from core enzyme after initiation
In summary:
- Core enzyme catalyzes RNA synthesis and elongation
- Sigma factor enables promoter recognition and initiation
- Together, they carry out the three stages of transcription
The sigma factor provides promoter specificity, while the core enzyme has the catalytic activity for RNA polymerization.
RNA polymerase
Consists of 5 subunits (2α, 1β, 1β’, 1ω)
Catalyzes RNA synthesis (nucleotide polymerization)
Responsible for elongation of RNA transcript
sigma factor
Dissociable subunit that binds core enzyme
Recognizes and binds specific promoter sequences
Enables core enzyme to bind promoters
Required for transcription initiation
Dissociates after initiation
holoenzyme
The complete transcription initiation complex
Consists of RNA polymerase core + sigma factor
Sigma factor confers promoter specificity
Holoenzyme binds promoter and initiates transcription
After initiation, sigma released and core does elongation
transcription initiation
Promoter Recognition:
Sigma factor of RNA polymerase holoenzyme recognizes and binds to promoter DNA sequence
Promoters have -10 and -35 regions that sigma factor binds to
Closed Complex Formation:
RNA polymerase holoenzyme binds to promoter, forming a stable closed complex
Open Complex Formation:
DNA strands are unwound/melted open around the transcription start site (+1)
Helped by sigma factor and transcription factors binding upstream
Initiation:
RNA polymerase begins synthesizing RNA transcript complementary to the coding strand
Incorporates first few ribonucleotides using DNA template strand
Forms an initiation complex or open complex
After initiation:
Sigma factor is released from the core RNA polymerase enzyme
Core enzyme proceeds into the elongation phase
Key players:
Promoter DNA sequence
Sigma factor for recognition
RNA polymerase holoenzyme
Transcription factors
The initiation phase sets up the transcription bubble and allows transition into elongation.
transcription
elongation
Here are the key points about the transcription elongation phase in bacteria:
Elongation Complex:
- After initiation, sigma factor dissociates
- Core RNA polymerase enters elongation phase as the elongation complex
RNA Synthesis:
- RNA polymerase moves along DNA template strand
- Adds ribonucleotides one by one to the 3’ end of growing RNA transcript
- Synthesizes in the 5’ to 3’ direction
- Unwinds DNA helix to expose template strand
Transcription Bubble:
- Region of unwound DNA around active site
- Maintains ~12-14 bp of DNA unwound
Proofreading:
- RNA polymerase has intrinsic 3’->5’ exonuclease activity
- Removes misincorporated ribonucleotides for high fidelity
Regulation:
- Elongation factors can modulate RNA polymerase’s activity
- Transcription-coupled repair mechanisms act during elongation
Termination Signals:
- Specific DNA sequences/structures signal termination
- Rho protein aids termination at some terminators
The elongation complex faithfully copies the DNA template into an RNA transcript until it reaches a termination signal.
transcription termination
Here are the key points about transcription termination in bacteria:
Intrinsic Termination:
- Occurs at specific DNA sequences that form a hairpin loop followed by a U-rich region
- Hairpin causes RNA polymerase to pause and dissociate from the DNA template
- Common in prokaryotes and some eukaryotes
Rho-dependent Termination:
- Involves the rho protein binding to the C-rich rho utilization (rut) site on the nascent RNA
- Rho translocates along the RNA, removing RNA polymerase from the DNA
- Common in prokaryotes
Steps:
1) Termination signal sequence is transcribed
2) RNA polymerase pauses/stalls
3) Conformational change destabilizes the transcription complex
4) Completed mRNA transcript is released
5) RNA polymerase dissociates from the DNA template
Terminators ensure efficient termination and recycling of RNA polymerase after genes are transcribed.
Termination prevents:
- Interference with downstream genes
- Depletion of RNA polymerase and ribonucleotide pools
Proper termination is critical for gene regulation and transcriptional control.
terminator sequence
DNA sequence that signals the end of transcription
Contains a hairpin loop followed by a U-rich region
Hairpin causes RNA polymerase to pause/stall
mRNA hairpin
Stem-loop structure formed by the nascent mRNA at the terminator
G-C base pairs in the stem, loop at the end
Hairpin causes physical disruption of transcription complex
Facilitates release of the mRNA transcript
Gene expression
is the process by which a gene gets turned on/off in a
cell to make RNA and proteins. Gene expression may be measured by
looking at the mRNA transcribed (mRNA expression), or the protein
translated from the mRNA (protein expression)
What binds to the promotor?
sigma factor
Where does RNAP begin transcribing?
TSS
The mRNA that would be produced from gene P above will only contain the coding sequence. T/F
F
Some parts of the mRNA will not be translated by the ribosome. T/F
T
If a repressor were bound to the silencer, would transcription likely occur? Y/N
N
- The terminator, silencer, and promoter are transcription factors. T/F
F
- General TF like sigma & activators are need to transcribe a gene at high levels. T/F
T
Bacterial Genomes
- 1 circular chromosome (& circular
plasmids) - Not-membrane bound in nucleus
(nucleoid region) - Double-stranded DNA
- Circular chromosome compacted nearly
1000x to fit in the nucleoid region - Bacterial histone-like proteins (bend
instead of wrap DNA)
Bacteria Genome Organization
- Smaller in size (#base pairs) than euk
- High proportion of protein-coding
sequence (only ~10% non-coding) - Coding sequences are continuous
(contiguous) *no introns - Both DNA strands can contain the
coding sequence
Operons
are clusters of genes transcribed as one unit. They have one
promoter and one mRNA is produced.
Each gene’s coding sequence is translated from the single mRNA. thrA, thrB,
and thrC proteins will be made from the single mRNA.
These proteins often function together in the cell.
Operons are common in bacteria and much less common in eukaryotes.
Inducible Operons
The lac operon genes will produce enzymes that break down lactose when
lactose is present.
Inducible operon is off until the inducer molecule ”induces” the operon by inhibiting
the repressor (through binding it) *Inhibit an inhibitor = ON
Repressible Operons
Tryptophan operon contains genes that work together to synthesize the amino acid tryptophan when
it is absent
Repressible operons are on until the co-repressor binds the repressor.
ligand
molecule that binds
a protein (e.g. Iron is the ligand
for Fur repressor)
Regulatory elements can be anywhere
The coding strand of a chromosome is not always the same strand of DNA
depending on which gene you are examining. T/F
T
- Bacteria DNA exists as chromatin. T/F
T
- To regulate the transcription of a gene, an enhancer, silencer, and operator cannot
all be used for the same gene. T/F
F
- Bacteria genes must exist in an operon. T/F
F
- Enhancer and silencer sequences can overlap with promoter sequences. T/F
T
- Silencers and enhancers can be upstream or downstream. T/F
T
Describe gene expression and understand why expression is a highly regulated process
- Gene expression is the process by which the instructions in a gene are used to synthesize functional gene products (proteins or non-coding RNAs)
- It involves two main steps:
- Transcription: DNA is transcribed into RNA
- Translation: RNA is translated into proteins
- Gene expression is highly regulated for several reasons:
- Different cells express different genes for specialized functions
- Genes are expressed at the right time and level based on the cell’s needs
- Regulating expression prevents wasteful overproduction of proteins
- It allows the cell to respond to environmental signals and changes
- Regulation occurs at multiple levels:
- Transcriptional regulation (activating/inhibiting transcription)
- RNA processing and stability
- Translational regulation
- Post-translational modifications of proteins
- Precise gene regulation is crucial for normal development, cellular differentiation, and maintaining proper cellular function
Describe the characteristics of bacteria genomes (contrasting with euk)
Bacterial Genomes:
- Single circular chromosome (some bacteria have additional small circular plasmids)
- Relatively small genome size (few million base pairs)
- Lack membrane-bound nucleus
- DNA is naked, not associated with histones
- Genes are densely packed with little non-coding DNA
- Operons allow coordinated regulation of related genes
- Absence of introns (genes are continuous)
- Rapid replication and cell division
Contrasting with Eukaryotic Genomes:
- Multiple linear chromosomes
- Much larger genome sizes (billions of base pairs)
- DNA packaged into nuclei with histones
- Significant amount of non-coding DNA (introns, regulatory regions)
- Genes are discontinuous (exons separated by introns)
- Complex transcriptional regulation at individual gene level
- Presence of membrane-bound organelles like nucleus, mitochondria
- Slower replication and cell cycle
Overall, bacterial genomes are simpler, more compact, and lack the complexity of eukaryotic regulation and nuclear organization.
Describe operons: inducible and repressible types
Operons:
- Operons are clusters of genes that are co-transcribed from a single promoter
- Allow coordinated regulation of related genes involved in the same pathway
- Characteristic of bacterial genomes and some archaea
Inducible Operons:
- Genes are normally turned off (not transcribed)
- Presence of an inducer molecule activates transcription
- Inducer binds to a repressor protein, inactivating it
- Allows RNA polymerase to bind and transcribe the operon
Example: lac operon in E. coli
- Induced by lactose to produce enzymes for lactose metabolism
- Lack of glucose also induces (glucose prevents induction)
Repressible Operons:
- Genes are constitutively transcribed by default
- Presence of a corepressor molecule represses/turns off transcription
- Corepressor binds to a repressor protein, activating its binding to operator
Example: trp operon in E. coli
- Repressed when tryptophan levels are high (corepressor present)
- Allows regulation of tryptophan biosynthesis
This coordinate regulation allows bacteria to efficiently express genes when needed and conserve resources otherwise.
Understand how both types of operons work and their regulatory elements &
transcription factors
co-repressors, repressors, activators, co-activators
Inducible Operons:
- Regulated by repressor protein that binds to operator, blocking transcription
- Inducer molecule binds to and inactivates repressor
- This allows RNA polymerase to bind promoter and transcribe operon
Repressible Operons:
- Regulated by repressor protein + corepressor molecule
- Corepressor binds repressor, increasing its affinity for operator
- Activates repressor binding, blocking RNA polymerase
Regulatory Elements:
- Promoter - RNA polymerase binding site to initiate transcription
- Operator - Repressor protein binding site that blocks transcription
- Inducer/Corepressor - Small molecules that modulate repressor activity
Transcription Factors:
- Repressors - Bind operator to repress transcription (e.g. LacI, TrpR)
- Activators - Bind DNA and recruit RNA polymerase (e.g. CRP for lac)
- Coactivators - Help activators bind/function (e.g. CAP for lac)
- Inducers - Inactivate repressors, allowing transcription
- Corepressors - Activate repressors to block transcription
This interplay between activators, repressors, inducers, and corepressors allows bacteria to precisely control operon expression in response to environmental/metabolic conditions.
Be able to recognize what type of operon regulation is occurring given
scenarios/information
Scenario 1:
Certain genes are normally not expressed. However, when a specific small molecule is added to the bacterial culture, those genes become expressed.
This describes an inducible operon. The small molecule acts as an inducer, inactivating the repressor protein and allowing transcription of the operon.
Scenario 2:
A set of enzymes involved in an amino acid biosynthesis pathway are produced continuously. But when the amino acid is added to the growth medium, production of those enzymes stops.
This is an example of a repressible operon. The amino acid acts as a corepressor, binding to and activating the repressor protein to block transcription of the operon.
Scenario 3:
Genes encoding enzymes for lactose metabolism are expressed only when lactose is present and glucose is absent in the growth medium.
This matches the regulation of the famous lac operon, which is an inducible operon. Lactose acts as the inducer, while glucose prevents induction (glucose repression).
Scenario 4:
A repressor protein is bound to the operator region, blocking transcription. Adding a specific metabolite causes the repressor to change shape and fall off the operator.
This scenario depicts an inducible operon where the metabolite is the inducer that inactivates the repressor protein bound at the operator.
The key is to identify if a small molecule is allowing transcription (inducer) or blocking it (corepressor) to distinguish inducible vs. repressible regulation.
Be able to read a transcriptional profile (gene expression data with the colored blocks)
for what is turned on/off
Here are some tips for reading a transcriptional profile or gene expression data represented by colored blocks to determine which genes are turned on or off:
- Look at the scale/legend to understand what the colors represent:
- Red typically indicates high expression/upregulation
- Green usually means low expression/downregulation
- Black or gray often represents baseline/no change
- Compare across conditions/time points:
- Bright red blocks indicate that gene is highly expressed in that condition
- Bright green blocks mean the gene is repressed/low expression
- Look for patterns within rows (same gene):
- A row with mostly red means that gene is generally highly expressed
- A row with mostly green suggests that gene has low expression levels
- Focus on differences between conditions within a row:
- A switch from red to green indicates the gene was downregulated
- Change from green to red means the gene became upregulated
- Subtle shades indicate degrees of change:
- Darker reds = very high expression
- Lighter greens = modest downregulation
- Black/gray blocks typically mean no significant change in expression.
The key is correlating the color intensity to expression levels based on the scale and looking for contrasting patterns between conditions/time points to identify up/downregulated genes. With practice, these visual profiles provide a powerful way to analyze gene expression data.
Understand that transcriptional regulation can occur in many combinations of
regulatory elements and transcription factors
- Transcriptional regulation is a complex process involving the interplay of multiple components:
- Regulatory DNA sequences (promoters, enhancers, silencers, insulators)
- Transcription factors (activators, repressors, co-regulators)
- Chromatin remodeling complexes
- DNA methylation/epigenetic modifications
- Promoters alone can have multiple binding sites for:
- Activators that recruit RNA polymerase
- Repressors that block polymerase binding
- General transcription factors required for initiation
- Enhancers are distal regulatory regions that can bind:
- Activators to increase transcription
- Repressors to decrease transcription
- Interactions with promoters via DNA looping
- Transcription factors can act cooperatively:
- Multiple activators synergize for stronger activation
- Activators and repressors compete for binding
- Co-activators/co-repressors modulate activity
- Chromatin structure and epigenetics add another layer:
- Open chromatin allows transcription factor access
- Closed/methylated chromatin blocks binding
- Chromatin remodelers alter accessibility
- Cell type and environmental signals dictate which factors are present
- The precise combination of sequences, binding factors, chromatin state determines the transcriptional output
This modularity and cooperativity of regulatory components allows incredibly precise spatiotemporal control of gene expression programs.
Another take away is that enhancers and silencers can overlap with promoters and
there is nothing that says enhancers have to be upstream and silencers downstream.
They can be either
histone-like proteins (I would say bacteria DNA is chromatin),
- Bacteria lack histones but have histone-like proteins called nucleoid-associated proteins (NAPs)
- NAPs help compact and organize bacterial DNA into a nucleoid structure
- The nucleoid functions like a simpler form of eukaryotic chromatin
- Examples of bacterial NAPs: HU, H-NS, IHF, Fis, Dps
- NAPs bind DNA, induce bends/loops, and condense the DNA molecule
- The bacterial nucleoid, organized by NAPs, is analogous to eukaryotic chromatin organized by histones
nucleoid-region
The nucleoid region is a condensed area within a prokaryotic cell where the genetic material, typically in the form of a circular chromosome, is located. It lacks a membrane-bound nucleus found in eukaryotic cells.
operon
An operon is a functional unit of genetic material found in prokaryotic cells consisting of a cluster of genes under the control of a single promoter. It includes structural genes that encode proteins, along with regulatory elements such as an operator and a promoter. The operon allows for coordinated gene expression, typically in response to environmental signals.
inducible operon
An inducible operon is a genetic regulatory system in prokaryotic cells where gene expression is normally off but can be turned on in response to specific environmental signals or molecules.
inducer molecule
An inducer molecule is a substance that initiates or enhances the expression of specific genes in an organism by interacting with regulatory proteins such as transcription factors.
repressible operon
A repressible operon is a genetic regulatory system in bacteria where the transcription of a group of genes is typically active, but can be inhibited (repressed) when a specific molecule, usually a product of the metabolic pathway controlled by those genes, is present in abundance.
co-repressor
A co-repressor is a molecule that binds to a repressor protein, enhancing its ability to inhibit the expression of specific genes. This binding typically occurs in regulatory systems where the presence of the co-repressor is associated with certain environmental or cellular conditions.
operator
An operator is a DNA sequence found within the promoter region of a gene that serves as a binding site for regulatory proteins, such as repressors or activators. The binding of these proteins to the operator can either inhibit or facilitate the initiation of transcription of the adjacent gene(s).
untranslated region (UTR)
The untranslated region (UTR) is a segment of mRNA (messenger RNA) that lies outside the coding sequence of a gene. It occurs both upstream (5’ UTR) and downstream (3’ UTR) of the coding region. While not translated into protein, UTRs play important roles in regulating gene expression, mRNA stability, and localization within the cell.
co-activator
A co-activator is a protein that interacts with transcription factors to enhance the transcriptional activity of specific genes. Co-activators typically do not directly bind to DNA but instead facilitate the assembly of transcriptional machinery or modify chromatin structure to promote gene expression. They play crucial roles in regulating various cellular processes by modulating gene transcription.
After you learn eukaryotic genomes and transcription be able to compare and contrast
bacteria and eukaryotic process
Similarities:
* Both involve proteins that bind and compact DNA
* Serve to organize and package the genetic material
* Help condense long DNA molecules into more compact structures
Differences:
* Eukaryotes use histone proteins, bacteria use histone-like NAPs
* Eukaryotic chromatin is more complex, with multiple levels of organization
* Bacterial nucleoid is a simpler, more loosely packaged structure
* Histones form nucleosomes, the basic unit of eukaryotic chromatin
* NAPs don’t form nucleosome-like structures in bacteria
* Chromatin undergoes more dynamic changes (condensation/decondensation) during cell cycle
* Bacterial nucleoid is more static, less regulated compaction
In essence:
* Both use proteins to compact and organize DNA
* But eukaryotic chromatin is a more complex, dynamic, and regulated process
* Bacterial nucleoid formation by NAPs is a simpler, more static process
Be able to do deduce from scenarios or diagrams what type of operon
(inducible/repressible) is being used or would be used. Like the iron-stealing operon example.
Here are some concise points to help deduce the type of operon (inducible or repressible) from scenarios or diagrams:
Inducible Operon:
- Genes are normally OFF or expressed at low levels
- Presence of an inducer molecule/signal turns ON expression
- Often used for catabolic operons (breaking down substrates)
- Example: lac operon induced by lactose, allows bacteria to metabolize lactose
Repressible Operon:
- Genes are normally ON or constitutively expressed
- Presence of a corepressor molecule/signal turns OFF expression
- Often used for anabolic operons (biosynthesis pathways)
- Example: trp operon repressed by tryptophan, stops production when enough tryptophan
Deducing from Scenarios/Diagrams:
- If operon is OFF normally, needs a molecule/signal to turn ON → Inducible
- If operon is ON normally, needs a molecule/signal to turn OFF → Repressible
- Look for inducers (substrates, metabolites) that activate expression → Inducible
- Look for corepressors (end-products) that repress expression → Repressible
- Iron-stealing operon likely inducible, turned ON by iron starvation signal
So analyze the normal/default state, regulatory molecules involved, and whether they activate or repress expression to deduce inducible vs repressible.
Be able to look at a transcriptional profile and understand how it shows genes being
turned on/off
- High signal/intensity = gene is expressed (turned on)
- Low/no signal = gene is not expressed (turned off)
- Compare conditions to see induction (turned on) or repression (turned off)
- Clustered patterns indicate co-regulated genes
- Sharp increases/decreases = genes turned on/off in response
- Consistent expression = constitutive (always on)
- Changes over time = dynamic regulation
- Correlate with known regulators/pathways for insights
Operon Summary
- Inducible operons are OFF (not transcribed) because repressor IS bound to the
operator in the absence of inducer molecule. Should make sense, operon off
without the inducer. - When inducer molecule present it binds to repressor and causes
repressor to dissociate from operator which induces the operon
(transcribed) - Repressible operons are ON (transcribed) because repressor is NOT bound to
the operator in the absence of co-repressor molecule. Should make sense,
operon on without the co-repressor. - When co-repressor molecule present it binds to repressor and causes
repressor to bind the operator which represses the operon (not
transcribed)
- Termination of transcription will happen:
b) somewhere soon after AAUAAA
Eukaryotic genomes
- Membrane-bound (nucleus)
chromosomes - Linear chromosomes
- Compacted and wrapped around
proteins called histones (8 histones
make up a nucleosome) - MOST compacted during
mitosis/meiosis - Lots of non-coding sequence (intergenic – between genes)
- AND non-coding sequence within genes (introns)
- Eukaryotic genes are NOT continguous; coding sequence (exons) is broken up with introns
- Genes go in both ‘directions’ – meaning the coding strand can be on the top or bottom DNA strand of the
double helix (same in bacteria) - Lots of repetitive sequences! (don’t worry about what theses are)
Post-transcriptional processing : mRNA modification
Eukaryotic mRNA made is in a pre-mRNA state – not actual message used to make
protein yet.
Pre-mRNA modified w/ Cap, polyA Tail, and spliced in the nucleus to become
mature mRNA!
Bacteria modify the 5’ end of
mRNAs too – but let’s just say it’s more
extensive in eukaryotes.
5’ cap - Modified guanine nucleotide added to 5’ of mRNA (technically happens
during txn). Function: bind ribosomes & protect mRNA from degradation
polyA tail
50-300 A’s; added after
termination. Function: protects from
degradation; Aids in nuclear export
of transcript
Post-transcriptional process: RNA splicing in eukaryotes
Notice there is an Un-Translated Region (UTR) both on the 5’ and 3’ end of the mRNA
Bacteria have UTRs as well.
Served regulatory functions as well.
Splicing technically happens co-/post-transcriptionally. Do
not worry about the specific order of processing like 5’ cap,
polyA, splicing. We will lump them as post-transcription.
Post-transcriptional regulation: Alternative splicing
MANY ways to splice mRNA
Each of these would
be translated into
a different protein!
from one MRNA
multiple variations
of the coding seq
that give unique
proteins.
Affinity & concentration
Different TFs (proteins) may also have different affinity for the same regulatory sequence.
Affinity is the strength/weakness at which an interaction occurs.
There may also be different amounts of the proteins available in the cell!
regulating seg. differences can affect TF
affinity.
Post-transcriptional regulation: RNA interference
Regulatory small non-coding RNAs (ncRNA) can regulate gene expression.
*usually bind in the 5’ or 3’ UTRs of mRNA transcripts; 20-25 nt long
short interfering RNA (siRNA) = perfect match – degrade mRNA (post-transcription reg)
micro RNA (miRNA) = imperfect match – block translation (post-transcription reg)
Bacteria do not have the proteins for siRNA but have ways to regulate their mRNAs post-
transcriptionally using the UTRs as well
Amino acid functional groups interact with major or minor
groove of DNA in sequence specific way
a) A-T, T-A, G-C, & C-G base pairs
present different shapes and chemical
groups in the grooves of DNA
Regulatory sequences are bound in a double-stranded
manner via a “hydrogen bonding” code in the major
and minor grooves
The same regulatory sequence can vary within genomes and be a
form of transcriptional regulation!
Bigger the letter the more often it is
found in the TATA box of the particular
type of genes.
Describe eukaryotic transcription steps initiation, elongation, and termination
Initiation:
RNA polymerase II binds to promoter region of gene
Transcription factors assist binding and initiation
General transcription factors recruit RNA pol II to promoter
TFIIH unwinds DNA to allow transcription bubble formation
Elongation:
RNA pol II moves along template strand, synthesizing RNA
Transcription elongation factors assist and regulate process
RNA pol II unwinds DNA ahead, re-anneals behind as it moves
5’ cap added to nascent mRNA
RNA splicing occurs - introns removed, exons joined
Termination:
Termination signals in DNA sequence trigger process
RNA pol II dissociates from DNA template
Polyadenylation factors add poly-A tail to 3’ end of mRNA
Fully processed, mature mRNA is released for translation
Transcription Initiation is always about recruiting RNAP to promoter
* Need TBP + general TFs to bind promoter = pre-initiation complex – how
is this different than bac?
* RNAP binds to pre-IC and all becomes the initiation complex
* Once initiation complex, RNAP unwinds DNA = initiation stage done
Recruiting RNA Polymerase II (RNAP II) to the promoter is key for initiation
TATA-binding protein (TBP) binds to TATA box in promoter
TBP assists binding of other general transcription factors (GTFs)
Together they form pre-initiation complex (pre-IC)
Difference from bacteria:
In bacteria, RNAP alone can bind promoter and initiate
In eukaryotes, RNAP II requires pre-IC with TBP and GTFs first
RNAP II joining pre-IC:
Pre-IC recruits RNAP II to promoter
RNAP II joins, forming full initiation complex (IC)
Initiation completion:
Within IC, RNAP II unwinds DNA around transcription start site
This formation of transcription bubble completes initiation stage
Understand DNA looping is one way distal elements can regulate transcription
initiation in both euk and bac
DNA Looping in Eukaryotes:
- Distal enhancers can be thousands of base pairs away from promoter
- DNA loops bring enhancers into close proximity with promoter
- This enhancer-promoter looping is mediated by transcription factors
- Allows regulatory proteins bound at enhancers to interact with transcription machinery
DNA Looping in Bacteria:
- Distal cis-regulatory sequences located upstream or downstream of promoter
- DNA is looped to allow binding of transcription factors near promoter
- Loop formation assisted by nucleoid-associated proteins
- Allows transcription factors bound at distant sites to influence initiation complex
General Points:
- DNA looping overcomes linear distance between regulatory elements and promoters
- Enables communication between distal binding sites and transcription machinery
- Allows integration of multiple regulatory signals for controlled initiation
Eukaryotic transcription elongation is just like in bacteria except bacteria
translate mRNA as it is being made. Euk cannot do this b/c transcription in
nucleus and translation in cytosol.
- The elongation process itself is similar in eukaryotes and bacteria
- RNA polymerase moves along DNA template, synthesizing mRNA
- Transcription elongation factors assist and regulate the process
- Key difference is the coupling to translation:
- In bacteria, translation can begin as mRNA is being transcribed (coupled)
- In eukaryotes, transcription occurs in nucleus but translation in cytoplasm
- So eukaryotic mRNA cannot be translated as it is being transcribed
- This separation is due to:
- Nuclear envelope barrier in eukaryotic cells
- mRNA must be fully transcribed, processed, exported to cytoplasm
- Only then can translation on mature mRNA occur in the cytoplasm
- In bacteria (no nuclear envelope):
- Ribosomes can directly access and translate the nascent mRNA
- Allowing co-transcriptional translation to occur efficiently
Termination uses a terminator sequence. In euk causes mRNA to be cut off and
released from RNAP. In euk right before terminator is a polyA signal which is
used post-transcriptionally to add polyA tail. How is this different than bac?
Eukaryotes:
- Termination guided by terminator sequence in DNA
- This causes RNAP to dissociate, releasing the nascent mRNA
- Just upstream of terminator is a poly(A) signal sequence
- This poly(A) signal is not used during transcription termination itself
- After termination, the poly(A) signal sequences guide:
- Endonucleolytic cleavage of the 3’ end of the mRNA
- Addition of a poly(A) tail to the 3’ end by poly(A) polymerase
Bacteria:
- Termination can occur through rho-independent or rho-dependent mechanisms
- Rho-independent uses RNA hairpin followed by U-rich sequence
- No poly(A) tail is added to bacterial mRNAs
- The 3’ end is simply the last transcribed portion
Key Differences:
- Eukaryotes use separate terminator and poly(A) signals
- Poly(A) tail added as a post-transcriptional processing step
- Bacteria lack poly(A) tails and processing of the 3’ end
- Termination signals are encoded differently
coding vs. non-coding sequences in genome (different than coding and template
strands!!)
Coding Sequences:
- Regions of DNA that get transcribed into mRNA
- The mRNA is then translated into proteins
- Include exons of genes that code for proteins
- Make up a small fraction of the genome (~1-2% in humans)
Non-Coding Sequences:
- Regions of DNA that do not code for proteins
- Not transcribed into mRNA that gets translated
- Include introns within genes
- Also regulatory regions like promoters, enhancers, silencers
- Repeat sequences, telomeres, centromeres etc.
- Comprise the vast majority of the genome (~98% in humans)
Key Points:
- Coding sequences are the protein-coding exons
- Non-coding includes introns, regulatory regions, other genomic sequences
- Non-coding sequences can still be transcribed into non-coding RNAs
- But they don’t get translated into proteins
- The non-coding portion is much larger than the coding portion
So in summary - coding = exons that get translated into proteins
- non-coding = introns, regulatory, other non-translated sequences
contiguous vs. non-contiguous genes
Here are the key points about contiguous versus non-contiguous genes:
Contiguous Genes:
- Genes that are located right next to each other on the same chromosome
- The coding sequences are contiguous/uninterrupted
- No non-coding regions or other genes in between
Non-Contiguous Genes:
- Genes that are separated by stretches of non-coding DNA
- The coding sequences are non-contiguous/interrupted
- Interspersed with introns, regulatory regions, other genes etc.
In both cases:
- The coding sequences (exons) are transcribed into mRNA
- Introns and other non-coding regions are removed during splicing
Key Differences:
- Contiguous genes don’t require splicing of the transcript
- Non-contiguous genes require splicing to remove interrupting sequences
- Contiguous genes are relatively rare in eukaryotes
- Most eukaryotic genes are non-contiguous due to presence of introns
So in summary:
- Contiguous = Coding sequences are together without interruptions
- Non-contiguous = Coding sequences are separated by non-coding regions
- Most eukaryotic genes are non-contiguous and require splicing
polyA signal
A polyadenylation signal, often abbreviated as polyA signal, is a specific nucleotide sequence in mRNA precursor molecules (pre-mRNA) that signals the addition of a polyadenylate (poly-A) tail during mRNA processing. This signal is recognized by proteins involved in mRNA maturation, leading to the cleavage of the pre-mRNA and the addition of a string of adenine nucleotides (poly-A tail) to the mRNA’s 3’ end. The poly-A tail plays roles in mRNA stability, transport, and translation.
RNA pol II vs I/III
RNA Polymerase II (RNA Pol II):
- Transcribes protein-coding genes
- Produces precursors of mRNA that will be translated into proteins
- Includes promoter recognition, initiation, elongation, termination
- Transcripts are capped, spliced, polyadenylated
RNA Polymerase I (RNA Pol I):
- Transcribes ribosomal RNA (rRNA) genes
- rRNA is a major constituent of ribosomes
- Found in nucleolus region of nucleus
RNA Polymerase III (RNA Pol III):
- Transcribes transfer RNA (tRNA) genes
- Also transcribes 5S rRNA, some small nuclear RNAs
- Transcripts are relatively short
Key Differences:
- RNA Pol II - mRNA transcription for protein synthesis
- RNA Pol I/III - transcription of non-coding RNAs
- Different promoter recognition and regulation mechanisms
- RNA Pol II transcripts are extensively processed
So in summary:
- RNA Pol II for protein-coding mRNAs
- RNA Pol I for large rRNA transcripts
- RNA Pol III for small stable RNAs like tRNAs
- Different roles but all essential for gene expression
TATA box
The TATA box, also known as the Goldberg-Hogness box, is a DNA sequence found upstream (usually around 25-30 base pairs upstream) of the transcription start site of many eukaryotic genes. It consists of the sequence “TATAAA” or a similar variant. The TATA box serves as a binding site for the TATA-binding protein (TBP), a component of the transcription factor IID (TFIID) complex. Binding of TBP to the TATA box helps to initiate the assembly of the RNA polymerase II transcription initiation complex, facilitating the start of transcription. The TATA box is crucial for the accurate positioning of the transcriptional machinery and the regulation of gene expression.
TATA
binding protein (TBP)
The TATA-binding protein (TBP) is a key component of the transcription factor IID (TFIID) complex in eukaryotes. It binds specifically to the TATA box, a DNA sequence found upstream of many eukaryotic genes, and plays a central role in the initiation of transcription by RNA polymerase II. TBP helps recruit other transcription factors and RNA polymerase II to the promoter region of genes, facilitating the assembly of the pre-initiation complex and the initiation of transcription. It is essential for the accurate regulation of gene expression.
general transcription factor
A general transcription factor is a protein involved in the initiation of transcription of protein-coding genes in eukaryotes. These factors are required for the assembly of the transcriptional machinery at the promoter region of genes. They include proteins like the TATA-binding protein (TBP) and other factors associated with RNA polymerase II. General transcription factors help to position RNA polymerase II correctly at the transcription start site and facilitate the initiation of transcription. They are essential for the transcription of most protein-coding genes and are distinct from regulatory transcription factors that bind to specific DNA sequences to modulate gene expression.
pre-initiation complex
The pre-initiation complex (PIC) is a multi-protein complex that forms at the promoter region of a gene during the initiation stage of transcription in eukaryotes. It includes general transcription factors, such as TATA-binding protein (TBP), and other proteins that help to position RNA polymerase II and initiate transcription. The assembly of the pre-initiation complex is a crucial step in gene expression, marking the beginning of transcription of a specific gene.
initiation complex
The initiation complex refers to the ensemble of proteins assembled at the promoter region of a gene to initiate transcription. In prokaryotes, the initiation complex typically involves RNA polymerase along with sigma factor, which helps recognize the promoter sequence. In eukaryotes, the initiation complex includes RNA polymerase II, general transcription factors (such as TATA-binding protein and TFIIA, TFIIB, etc.), and other regulatory proteins. The formation of the initiation complex marks the beginning of transcription and is a key step in gene expression.
Describe eukaryotic RNA post-transcriptional processing: 5’ cap, 3’ polyA tail, splicing
* know functions of 5’ cap and 3’ polyA tail
5’ Capping:
- 7-methylguanosine cap added to 5’ end of nascent mRNA
- Functions:
- Protects mRNA from degradation
- Assists nuclear export of mRNA
- Recruits ribosomes for translation initiation
3’ Polyadenylation:
- Poly(A) tail of ~200 adenines added to 3’ end
- Functions:
- Protects mRNA from degradation
- Facilitates transport from nucleus
- Enhances translation by stabilizing mRNA
Splicing:
- Removal of non-coding intron sequences from pre-mRNA
- Joining of coding exon sequences into mature mRNA
- Occurs in nucleus before mRNA export
- Requires spliceosome complex of snRNPs and proteins
Additional points:
- All three processes occur in nucleus before export
- Allow proper maturation and regulation of eukaryotic mRNAs
- Increase stability, translation efficiency of mature transcripts
- Splicing enables combination of exons, protein diversity
Describe alternative splicing in eukaryotes
* alt. splicing as a form of post-transcriptional regulation
- Most eukaryotic genes contain multiple exons and introns
- During splicing, different combinations of exons can be included in the mature mRNA
- This produces multiple different mRNA transcripts from the same gene
- Mechanisms of alternative splicing:
- Exon skipping - certain exons are omitted
- Mutually exclusive exons - only one of two exons is included
- Alternative 5’ or 3’ splice sites are used
- Intron retention - an intron may remain in the mRNA
- Allows a single gene to code for multiple different protein isoforms
- Expands the protein diversity encoded by the genome
- Alternative splicing is tightly regulated
- By splicing regulatory proteins
- Binding of these proteins influences exon inclusion
- Allows splicing to respond to developmental/environmental cues
- Functions as a form of post-transcriptional gene regulation
- Controls which protein isoforms are produced
- Modulates protein function, localization, interactions
- Very prevalent in humans, contributes to protein diversity
- Up to 95% of human genes may undergo alternative splicing
So in summary, alternative splicing is a crucial mechanism that allows production of multiple protein variants from one gene through differential exon inclusion.
Describe RNA interference and two ways it regulates gene expression
* siRNA and mRNA stability – post transcriptional regulation
* miRNA and translation inhibition – post-transcriptional regulation
- siRNA and mRNA Stability (Post-Transcriptional Regulation):
- siRNAs (small interfering RNAs) are ~20 nucleotides long
- Derived from long double-stranded RNA precursors
- The siRNA associates with the RISC complex
- RISC uses siRNA as a guide to bind and cleave complementary mRNA targets
- This degradation of the mRNA prevents its translation into protein
- Acts as a post-transcriptional gene silencing mechanism - miRNA and Translation Inhibition (Post-Transcriptional Regulation):
- miRNAs (microRNAs) are also ~22 nucleotides long
- Derived from stem-loop precursors encoded in the genome
- The miRNA associates with the RISC complex
- RISC uses the miRNA to bind to partially complementary sites in 3’UTRs of mRNAs
- This does not degrade the mRNA, but represses its translation into protein
- Allows miRNAs to fine-tune protein output post-transcriptionally
In both cases:
- RNAi pathways involve small non-coding RNAs
- These guide Argonaute-containing RISC complex to targets
- Results in post-transcriptional regulation of gene expression
- siRNAs degrade mRNAs, miRNAs inhibit translation without degradation
So in summary, RNAi exploits small RNAs to regulate genes post-transcriptionally by either mRNA degradation (siRNA) or translational repression (miRNA).
Describe where and how DNA binding proteins recognize specific DNA sequences (both
bacteria & eukarya)
Bacteria:
- Many bacterial transcription factors contain helix-turn-helix motifs
- The α-helices in this motif fit into the major groove of DNA
- Specific amino acids make contacts with exposed DNA bases
- This allows sequence-specific recognition of promoter regions
Eukaryotes:
- Eukaryotic transcription factors use several DNA binding motifs:
- Zinc finger proteins - zinc ions help position α-helices in DNA major groove
- Leucine zipper - dimerization allows recognition of longer sequences
- Helix-loop-helix - HLH motifs bind DNA and allow dimerization
- Other motifs like homeodomain, HMG box also facilitate DNA binding
General Mechanisms:
- α-Helices in the protein motifs are key for DNA major groove binding
- Amino acid side chains make sequence-specific contacts with exposed bases
- Dimerization of DNA binding domains increases binding specificity
- Minor groove interactions and DNA bending also contribute
Additional Points:
- Cooperative binding with other proteins enhances specificity
- Post-translational modifications can modulate DNA binding affinity
- Chromatin structure impacts accessibility of binding sites in eukaryotes
So in essence, DNA binding proteins use a variety of structural motifs that allow their α-helices and amino acids to specifically read out and bind target DNA sequences.
Understand how regulatory sequence variation and TF affinity and concentration can be
used to regulate transcription initiation. (both bacteria & eukarya)
Regulatory Sequence Variation:
- Mutations in promoter or enhancer/operator sequences
- Can increase or decrease affinity for TF binding
- Alters recruitment of transcription machinery and initiation frequency
TF Affinity:
- Determined by TF structure and DNA sequence motif
- Higher affinity allows tighter TF binding to regulatory regions
- Increases probability of initiation complex assembly
TF Concentration:
- Abundance of specific TFs is a major determinant
- Higher TF levels increase occupancy at regulatory sequences
- More efficient recruitment of polymerase for initiation
In Bacteria:
- Promoter mutations affect binding of σ factors and RNAP
- Operator mutations modulate repressor/activator binding
- TF levels controlled by regulation of their expression
In Eukaryotes:
- Promoter and enhancer mutations impact TF and coactivator binding
- Variation in activation domains affects recruitment ability
- TF levels controlled by complex regulatory mechanisms
So in both systems, changes to regulatory sequences, TF-DNA binding affinities, and cellular TF concentrations can all be leveraged to fine-tune transcription initiation rates for different genes under different conditions.
splicing
Splicing is the process in eukaryotic cells by which introns, non-coding regions of a gene, are removed from the precursor messenger RNA (pre-mRNA) transcript and the exons, coding regions, are joined together to form mature mRNA. This process occurs in the cell nucleus and is catalyzed by the spliceosome, a complex of RNA and protein. Splicing is essential for generating functional mRNA transcripts that can be translated into proteins.
alternative splicing
Alternative splicing is a process in eukaryotic gene expression where different combinations of exons within a pre-mRNA transcript can be joined together during splicing, resulting in multiple mRNA isoforms derived from a single gene. This process allows for the production of diverse protein products from a limited number of genes and contributes to cellular diversity and complexity. Alternative splicing can generate mRNA transcripts with different coding sequences, leading to proteins with distinct functions, localization, or regulatory properties.
intron
An intron is a non-coding segment of a gene’s DNA that is transcribed into pre-mRNA but is removed during RNA processing and does not appear in the final mRNA molecule. In eukaryotic cells, introns interrupt the coding sequences (exons) of genes and are spliced out during mRNA maturation. Although they do not encode protein sequences, introns can have regulatory functions and contribute to genetic diversity through alternative splicing.
exon
An exon is a coding region of a gene’s DNA that is transcribed into mRNA and appears in the final, mature mRNA molecule after introns have been removed during RNA processing. Exons contain sequences that encode specific amino acids, and they are joined together during splicing to form the coding sequence of the mRNA. Exons determine the protein-coding information of a gene and are crucial for synthesizing functional proteins.
UTR
The untranslated region (UTR) is a segment of mRNA (messenger RNA) that lies outside the coding sequence of a gene. It occurs both upstream (5’ UTR) and downstream (3’ UTR) of the coding region. While not translated into protein, UTRs play important roles in regulating gene expression, mRNA stability, and localization within the cell.
pre-mRNA
Pre-mRNA, or precursor messenger RNA, is an initial transcript produced during transcription in eukaryotic cells. It contains both exons (coding regions) and introns (non-coding regions). Pre-mRNA undergoes processing, including capping, splicing, and polyadenylation, to become mature mRNA. This processing involves the removal of introns and the addition of a 5’ cap and a poly(A) tail. The mature mRNA is then transported to the cytoplasm for translation into protein.
mature mRNA
Mature mRNA is the final processed form of mRNA that is ready for translation into protein in eukaryotic cells. It is derived from pre-mRNA through processing steps including capping, splicing, and polyadenylation. Mature mRNA contains only the exons (coding regions) of the gene and has a 5’ cap structure and a poly(A) tail added to it. It serves as the template for protein synthesis during translation.
5’ G-cap
The 5’ G-cap, also known as the 5’ cap, is a modified nucleotide structure added to the 5’ end of eukaryotic mRNA molecules during RNA processing. It consists of a guanine nucleotide linked to the mRNA via a 5’-5’ triphosphate bridge, followed by additional methylations. The 5’ cap plays crucial roles in mRNA stability, translation initiation, and mRNA export from the nucleus to the cytoplasm.
polyA tail
The poly(A) tail is a stretch of adenine nucleotides added to the 3’ end of eukaryotic mRNA molecules during RNA processing. It typically consists of multiple adenine residues, forming a “tail” structure. The poly(A) tail plays important roles in mRNA stability, facilitating mRNA export from the nucleus, and enhancing translation efficiency. It also protects the mRNA from degradation by exonucleases.
RNA interference
RNA interference (RNAi) is a biological process in which small RNA molecules, such as small interfering RNA (siRNA) or microRNA (miRNA), regulate the expression of genes by inhibiting the translation of mRNA or by promoting the degradation of specific mRNA molecules. RNAi acts as a natural defense mechanism against viruses and transposons and also plays essential roles in regulating gene expression during development and in response to environmental stimuli.
siRNA
Short interfering RNA (siRNA) is a class of small RNA molecules typically around 20-25 nucleotides in length. siRNAs are involved in the RNA interference (RNAi) pathway, where they mediate sequence-specific gene silencing by targeting complementary mRNA molecules for degradation or by inhibiting their translation. In experimental settings, synthetic siRNAs can be introduced into cells to specifically silence target genes, making them powerful tools for studying gene function and potential therapeutic agents for treating diseases caused by aberrant gene expression.
miRNA
MicroRNA (miRNA) is a class of small non-coding RNA molecules typically around 21-25 nucleotides in length. miRNAs are involved in post-transcriptional regulation of gene expression by binding to complementary sequences in the 3’ untranslated regions (UTRs) of target mRNA molecules. This binding can lead to mRNA degradation or inhibition of translation, thereby modulating the expression of target genes. miRNAs play critical roles in various biological processes, including development, differentiation, proliferation, and apoptosis, and dysregulation of miRNA expression has been implicated in many diseases, including cancer and neurodegenerative disorders.
major groove
The major groove is one of the two larger grooves found in the double helical structure of DNA, the other being the minor groove. It is characterized by a wider space between the two strands of the DNA helix and provides access to the nitrogenous bases of the nucleotides. The major groove is important for interactions with proteins, such as transcription factors and other DNA-binding proteins, which recognize specific sequences of DNA bases by fitting into and making contacts within this groove. It plays a crucial role in various cellular processes, including gene regulation, replication, and repair.
minor groove
The minor groove is one of the two smaller grooves found in the double helical structure of DNA, the other being the major groove. It is characterized by a narrower space between the two strands of the DNA helix and provides access to the edges of the nitrogenous bases of the nucleotides. While not as spacious as the major groove, the minor groove still plays a significant role in DNA-protein interactions, as certain DNA-binding proteins can recognize specific DNA sequences by fitting into and making contacts within this groove. The minor groove is also involved in various cellular processes, including gene regulation, replication, and repair.
affinity
Affinity refers to the strength of interaction between two molecules, such as a ligand and a receptor, or an enzyme and its substrate. It quantifies how readily molecules bind to each other and is typically measured by the equilibrium binding constant (Kd) in biochemistry. High affinity indicates a strong binding interaction, with molecules staying bound for longer periods or requiring lower concentrations for binding, whereas low affinity indicates weaker binding, with molecules dissociating more readily or requiring higher concentrations for binding. Affinity plays a crucial role in various biological processes, including signal transduction, enzyme catalysis, and molecular recognition.
Bacteria vs eukaryotic
Promoter
RNA Pol(s)
General TFs
Enhancers/silencers
Activators/repressors
Transcription Elongation
Transcription Termination
Post-transcriptional
5’/3’ UTR
Genome feature differences
Chromosome shape
Gene structure
Bacteria:
- Promoters for RNAP binding/initiation
- Single RNAP enzyme
- Sigma factors act as general TFs
- No enhancers/silencers
- Activators and repressors control transcription
- Transcription elongation by RNAP alone
- Rho-dependent or -independent termination
- No post-transcriptional modifications like capping/polyadenylation
- 5’/3’ UTRs present but no splicing
Eukaryotes:
- Promoters with TF binding sites
- 3 RNAP enzymes (I, II, III)
- Multiple general TFs like TFIID
- Enhancers and silencers regulate genes
- Activators, repressors, chromatin factors
- Transcription elongation involves many factors
- Termination with poly(A) site, processing factors
- 5’ capping, 3’ polyadenylation, splicing of introns
- Long 5’/3’ UTRs with regulatory roles
Genome Features:
- Bacterial genome is circular chromosome
- Eukaryotic genome has linear chromosomes
- Bacterial genes are simple, contiguous
- Eukaryotic genes have split exon/intron structure
In general, eukaryotic transcription has more protein factors involved and additional regulatory complexity from chromatin, RNA processing, and genome organization compared to the streamlined bacterial system.
Understand transcription and gene regulation in the context of chromatin
* Describe how heterochromatin vs. euchromatin controls gene expression
* Describe which parts of DNA would be more accessible – linker DNA vs.
nucleosomal DNA
Heterochromatin vs Euchromatin:
- Heterochromatin is densely packed, transcriptionally inactive
- Euchromatin is more open and accessible for transcription
- Heterochromatin limits binding of transcription factors/machinery
- Euchromatin allows transcriptional machinery access to DNA
- Highly condensed heterochromatin marks genes for silencing
- More open euchromatin structure permits active transcription
Nucleosomal DNA vs Linker DNA:
- Nucleosomes wrap DNA around histone octamers
- The linker DNA between nucleosomes is more exposed
- Transcription factors/complexes can more easily access linker DNA
- Nucleosomal DNA is less accessible when wrapped on histones
- Nucleosome positioning/remodeling exposes or hides sequences
- Activating sequences preferentially located in linker regions
Additional Regulation:
- Histone modifications (acetylation, methylation) affect chromatin
- ATP-dependent remodeling complexes alter nucleosome positioning
- Higher order folding into chromatin fibers also impacts accessibility
In summary:
- Heterochromatin is restrictive while euchromatin permits transcription
- Linker DNA is more accessible than nucleosomal DNA for binding
- Chromatin remodeling and histone modifications regulate accessibility
So the chromatin environment acts as a major regulator of transcription through modulating the physical accessibility of DNA sequences.
Describe epigenetics
* Understand how histone modifications regulate gene expression via chromatin
remodeling
* Describe epigenetic inheritance
Histone Modifications and Chromatin Remodeling:
- Histones can be covalently modified (e.g. acetylation, methylation)
- These modifications alter the charge and structure of nucleosomes
- Affect how tightly DNA is wrapped around histones
- Recruit or prevent binding of chromatin remodelers/transcription factors
- Histone acetylation generally leads to more open, transcriptionally active chromatin
- Histone methylation can lead to active or inactive states depending on residue
- ATP-dependent chromatin remodeling complexes can:
- Slide or remove nucleosomes to expose DNA sequences
- Regulate access of transcription machinery to genes
- Histone modifications and chromatin remodeling control gene expression epigenetically
Epigenetic Inheritance:
- Epigenetic marks like DNA methylation and histone modifications are mitotically inherited
- Patterns are propagated through cell divisions by enzymes
- Allows stable propagation of gene expression states
- In some cases, epigenetic states can be inherited transgenerationally
- Allows transmission of environmentally-induced epi-mutations
So in summary:
- Epigenetics refers to heritable modifications that regulate gene expression
- Without altering the underlying DNA sequence itself
- Histone modifications and chromatin remodeling are key epigenetic mechanisms
- Epigenetic information can be mitotically and sometimes transgenerationally inherited
euchromatin
Euchromatin is a loosely packed form of chromatin found in the nucleus of eukaryotic cells. It is characterized by a more open and accessible structure, allowing for active gene transcription and gene expression. Euchromatin is typically associated with genes that are actively transcribed to produce RNA, and it contains a higher density of genes and regulatory elements compared to heterochromatin. The less condensed nature of euchromatin facilitates the binding of transcription factors and other regulatory proteins to DNA, enabling efficient gene expression.
heterochromatin
Heterochromatin is a densely packed form of chromatin found in the nucleus of eukaryotic cells. It is characterized by a tightly condensed structure, which makes it less accessible for gene transcription and gene expression. Heterochromatin is typically associated with regions of the genome that are transcriptionally inactive or contain repetitive DNA sequences, such as centromeres and telomeres. The condensed nature of heterochromatin prevents the binding of transcription factors and other regulatory proteins to DNA, thereby silencing gene expression in these regions.
nucleosome
A nucleosome is the fundamental unit of chromatin, the complex of DNA and proteins that make up chromosomes in eukaryotic cells. It consists of a segment of DNA wrapped around a core of histone proteins. The core histones, comprising two copies each of histones H2A, H2B, H3, and H4, form an octamer around which approximately 147 base pairs of DNA are wound in about 1.65 turns. Nucleosomes serve to compact and organize the long strands of DNA within the cell nucleus and play crucial roles in gene regulation, DNA replication, and repair.
histone
Histones are a family of highly conserved proteins found in eukaryotic cell nuclei. They are responsible for packaging and organizing DNA into structural units called nucleosomes, which form the building blocks of chromatin. Histones play a crucial role in regulating gene expression by controlling access to DNA and influencing chromatin structure. They have a high density of positively charged amino acids, allowing them to interact with the negatively charged phosphate backbone of DNA. In addition to their structural role, histones can undergo various post-translational modifications, such as acetylation, methylation, and phosphorylation, which further regulate chromatin structure and gene expression.
nucleosomal DNA
Nucleosomal DNA refers to the DNA segment that is wrapped around the histone octamer core to form a nucleosome. It consists of approximately 147 base pairs of DNA that are wound around the histone proteins in about 1.65 turns. Nucleosomal DNA plays a crucial role in the organization and compaction of chromatin structure within the cell nucleus. Additionally, it is subject to various epigenetic modifications and influences gene expression by regulating the accessibility of DNA to transcription factors and other regulatory proteins.
linker
DNA
Linker DNA, also known as linker histone, refers to the DNA segment that connects adjacent nucleosomes in chromatin. It is the stretch of DNA that extends between two nucleosomes and is not tightly wrapped around histone proteins. Linker DNA contributes to the higher-order organization of chromatin by providing flexibility and allowing nucleosomes to be spaced apart. It also plays a role in regulating chromatin structure and gene expression by influencing the accessibility of DNA to regulatory proteins.
condensed chromatin
Condensed chromatin refers to regions of chromatin that are tightly packed and highly compacted within the cell nucleus. It is characterized by a dense arrangement of nucleosomes and other associated proteins, resulting in limited accessibility of the DNA to transcription factors and other regulatory proteins. Condensed chromatin is typically transcriptionally inactive and is often associated with heterochromatic regions of the genome, such as centromeres and telomeres. The compaction of chromatin into condensed structures helps to organize and protect the genome and plays a role in regulating gene expression and other nuclear processes.
decondensed chromatin
Decondensed chromatin refers to regions of chromatin that are less compacted and more open within the cell nucleus compared to condensed chromatin. It is characterized by a looser arrangement of nucleosomes and other associated proteins, allowing for increased accessibility of the DNA to transcription factors and other regulatory proteins. Decondensed chromatin is often associated with transcriptionally active regions of the genome, where genes are actively transcribed to produce RNA. The relaxation of chromatin structure into a decondensed state facilitates gene expression and other nuclear processes such as DNA replication and repair.
methylation
Methylation is a biochemical process where a methyl group (CH3) is added to a molecule, typically occurring on DNA, RNA, proteins, or small molecules. In the context of DNA, DNA methylation involves the addition of a methyl group to the cytosine base of DNA, typically at cytosine-phosphate-guanine (CpG) dinucleotide sites. DNA methylation plays a crucial role in regulating gene expression, genome stability, and chromatin structure. In gene regulation, methylation of CpG islands in gene promoter regions can lead to transcriptional repression by inhibiting the binding of transcription factors or recruiting proteins involved in gene silencing. In addition to DNA, methylation can also occur on histone proteins and RNA molecules, influencing various cellular processes and gene expression patterns.
acetylation
Acetylation is a biochemical process in which an acetyl group (-COCH3) is added to a molecule, typically occurring on proteins or histone proteins. In the context of histone proteins, acetylation involves the addition of an acetyl group to the lysine residues of histone tails. This modification is catalyzed by histone acetyltransferase (HAT) enzymes. Histone acetylation is associated with relaxed chromatin structure and increased gene expression, as it neutralizes the positive charge of lysine residues, weakening the interaction between histones and DNA, and facilitating access of transcription factors to DNA. Acetylation of non-histone proteins, such as transcription factors, can also regulate their activity and stability, influencing gene expression and various cellular processes.
chromatin
remodeling
Chromatin remodeling refers to the dynamic process by which the structure of chromatin, the complex of DNA and proteins in the nucleus of eukaryotic cells, is altered to regulate gene expression and other nuclear processes. It involves the repositioning, eviction, or restructuring of nucleosomes along the DNA, thereby modulating the accessibility of DNA to transcription factors and other regulatory proteins. Chromatin remodeling complexes, such as ATP-dependent chromatin remodelers, use energy from ATP hydrolysis to drive the movement or restructuring of nucleosomes, allowing for changes in chromatin organization and gene regulation. Chromatin remodeling plays crucial roles in diverse biological processes, including transcriptional activation and repression, DNA repair, and genome stability.
HAT
HAT stands for Histone Acetyltransferase. It is an enzyme that catalyzes the addition of an acetyl group (-COCH3) to lysine residues on histone proteins. This process, known as histone acetylation, typically occurs on the N-terminal tails of histones and is associated with relaxed chromatin structure and increased gene expression. HAT enzymes play a crucial role in regulating gene expression by modifying chromatin structure, making DNA more accessible to transcription factors and other regulatory proteins. They are involved in various cellular processes, including development, differentiation, and response to environmental cues.
HDAC
HDAC stands for Histone Deacetylase. It is an enzyme that catalyzes the removal of acetyl groups from lysine residues on histone proteins. This process, known as histone deacetylation, typically results in a more condensed chromatin structure and decreased gene expression. HDAC enzymes play a crucial role in gene regulation by reversing the effects of histone acetylation, leading to transcriptional repression. They are involved in various cellular processes, including development, differentiation, and response to environmental cues. HDAC inhibitors have been studied as potential therapeutics for various diseases, including cancer and neurodegenerative disorders, due to their ability to modulate gene expression patterns.
HMT
HMT stands for Histone Methyltransferase. It is an enzyme responsible for catalyzing the addition of methyl groups (-CH3) to lysine or arginine residues on histone proteins. This process, known as histone methylation, can result in different outcomes depending on the specific amino acid residue modified and the number of methyl groups added. Histone methylation can either activate or repress gene expression, depending on the site and degree of methylation, and is involved in regulating various cellular processes, including development, differentiation, and disease. HMTs play crucial roles in establishing and maintaining histone methylation patterns, which contribute to the epigenetic regulation of gene expression.
what processes of transcription
would be impossible if the gene above were in the heterochromatin state?
a) transcription factor binding to regulatory elements for initiation
b) elongation
What state of chromatin will transcribed genes most likely be in then?
b) euchromatin
DM
demethylases (removes)
Bacteria have compacted chromosomes as well and also histone-like proteins
but they do not use this. process to regulate it (as far as I know)
DNA can also directly be methylated and is another
form of epigenetics
Summary of regulatory points
Transcription (all at initiation)
- chromatin accessibility of the gene
*euk only (though bac do have
”chromatin”)
- DNA methylation *both
- activators, repressors, silencers,
enhancers, operators *both
Post-transcriptional
- alternative splicing of mRNA *euk only
- mRNA modification *both
- but polyA and 5’cap are *euk only
-5’ and 3’ UTR regulation *both
- small nc-RNAs binding to UTRs *both
but siRNA only euk.
Know which histone modifications and how they affect the accessibility of DNA
sequences.
***Really good to have big picture synthesis and connection of all parts of gene expression and
its regulation so far and how each step happens (process) and points of control (regulation) at
each step.
Transcription Initiation:
- Regulated by promoter sequences and bound transcription factors
- Chromatin accessibility controlled by histone modifications/remodeling
- e.g. H3K9ac increases accessibility, H3K9me3 decreases accessibility
- DNA looping brings distal enhancers/silencers into proximity of promoters
- Concentrations and activities of transcription factors modulate initiation
Elongation:
- Regulated by elongation factors and chromatin structure
- Histone modifications affect elongation efficiency
- e.g. H3K36me3 prevents initiation in gene bodies
- Chromatin remodelers clear nucleosomes for polymerase progression
Termination:
- Regulated by terminator sequences, poly(A) signals
- Proper chromatin context required for termination factors to bind
RNA Processing:
- 5’ capping, 3’ polyadenylation, splicing are regulated
- Splicing regulated by splicing factors binding signals in RNA
- Alternative splicing expands protein diversity
Epigenetic Regulation:
- Histone modifications and DNA methylation control chromatin state
- Inherited patterns maintain active/repressed expression states
- Chromatin remodelers expose or conceal regulatory regions
So in summary, every stage of eukaryotic gene expression is subject to multiple layers of regulation, with chromatin structure and epigenetic marks playing a central role in modulating accessibility and recruitment of the required machinery.
Know the features of the genetic code and what they mean – triplet nucleotide codon,
redundancy, NOT ambiguous, universality, wobble position, reading frame, and types of point
mutations (missense, nonsense, silent, and frameshift)
Triplet Nucleotide Codons:
- The genetic code is read in triplets of 3 nucleotides (codons)
- Each codon specifies a particular amino acid or stop signal
Redundancy:
- Multiple codons can code for the same amino acid
- This redundancy is described as the code being degenerate
Non-ambiguity:
- Each codon specifies only one amino acid (or stop)
- The code has no ambiguity in its meanings
Universality:
- The genetic code is nearly universal across all living organisms
- A few exceptions in certain viruses/prokaryotes
Wobble Position:
- The third position of a codon is more flexible in base-pairing
- Allowssome non-canonical base pairings (wobble)
Reading Frame:
- The codons are read in a continuous sequence without overlaps
- Frameshifts result in completely different proteins
Point Mutations:
- Missense - Single nucleotide change results in different amino acid
- Nonsense - Single nucleotide change introduces premature stop codon
- Silent - Single nucleotide change has no effect on amino acid
- Frameshift - Insertion/deletion shifts reading frame of all codons after
So in summary: Triplet codons, redundancy, non-ambiguity, near universality, wobble in third position, maintenance of continuous reading frame, and potential for different point mutation types are key features of the genetic code.
Even if activators and repressors are present, they may not be able to bind to their
regulatory elements depending on chromatin state. T/F
T
Even in the euchromatin state, nucleosomes have to be temporarily removed for
transcription elongation to occur. T/F
T
Heterochromatin is the compacted state of chromatin and histone acetylation causes
heterochromatin to form. T/F
F
What proteins are required to convert heterochromatin to euchromatin?
demethylase and acetyltransferase
We would expect an actively transcribed gene to be in a heterochromatin state. T/F
F
Epigenetic changes are a normal cellular process but can be caused by environmental
exposures. T/F
T
Epigenetic changes can occur at the histone and DNA level. T/F
T
Different cell types like skin vs. brain cells express different genes. One way to produce
this is to remodel chromatin such that skin specific genes are in euchromatin and other cell-specific genes in heterochromatin. T/F
T
Different cell types like skin vs. brain cells express different genes. One way to produce
this is to remodel chromatin such that skin specific genes are in euchromatin and other cell-
specific genes in heterochromatin. T/F
T
Different cell types like skin vs. brain cells express different genes. One way to produce a
skin cell is to express skin-specific transcription factors in skin cells only. T/F
T
