Lecture 40
Page 1: Lecture Information
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Final Exam Details:
The final exam will consist of approximately 70 questions.
About 50% of the material will cover new concepts presented from Lecture 27 onwards, while the remaining 50% will include material reviewed from previous exams (Exams 1 and 2).
Page 2: Weight Loss Challenges
In the News:
Recent discussions highlight the yo-yo effect observed in weight loss, where individuals manage to lose weight but subsequently struggle significantly to maintain it over time.
A study has centered on the roles of transcription and epigenetics in both humans and mice, examining biological responses before and after the weight loss process.
Page 3: Epigenetics and Weight Loss
Research Findings:
Individuals who underwent bariatric surgery and successfully reduced their Body Mass Index (BMI) by over 25% exhibited lasting transcriptional changes, especially in adipocytes (fat cells).
Similar trends were recorded in mice; these subjects regressed to their original weight significantly faster compared to control mice, suggesting lasting impacts from the initial weight loss.
The concept of "epigenetic memory" is crucial here, indicating that genes can retain health patterns and information prior to significant weight loss.
The findings imply a strong epigenetic component influencing the difficulties associated with sustaining weight loss, opening discussions on potential epigenetic therapies that could aid in maintaining weight loss.
Page 4: Introduction to Transcription
Definition:
Transcription is the biological process where RNA is synthesized from a DNA template. It plays a fundamental role in gene expression and regulation.
Page 5: RNA and Protein Synthesis
Genetic Flow:
The fundamental flow of genetic information can be summarized as follows: DNA → RNA → Protein.
Role of RNA:
It is essential to note that not all RNA molecules synthesized are translated into proteins; many RNA types function independently or regulate other molecules.
Through processing, assembly, inhibition, and degradation, RNA fulfills other regulatory roles critical for cellular functions.
Page 6: Comparison: Transcription vs DNA Replication
Similarities:
During both processes, RNA is synthesized in the 5' to 3' direction, and energy is also supplied from triphosphate nucleotides (rNTPs).
Both processes require a single strand of DNA as a template, necessitating unwinding of the DNA double helix.
Differences:
Transcription utilizes RNA nucleotides, whereas DNA replication employs DNA nucleotides.
Unlike DNA synthesis, transcription does not require a primer.
Only one DNA strand serves as the template for transcription, focusing on a single or few genes, contrasting with the genome-wide copying in replication.
The resultant product of transcription is single-stranded RNA, whereas DNA replication results in double-stranded DNA.
The enzyme responsible for transcription is RNA polymerase, not DNA polymerase.
Transcription may occur continuously, while DNA synthesis typically takes place at specific periods within the cell cycle.
Page 7: Key Differences
Detailed Differences Between Transcription and DNA Synthesis:
Transcription uses RNA nucleotides instead of DNA nucleotides.
RNA polymerase operates independently, without a prerequisite for a primer.
A single DNA strand is used for transcription, allowing for localization to one or a few specific genes rather than across entire chromosomes.
The outcome of transcription is single-stranded RNA, contrasting with the double-stranded nature of DNA produced during synthesis.
Importantly, transcription is regularly occurring within cells, while DNA replication primarily happens during the S phase of the cell cycle.
Page 8: Visualization
Visual Aid:
For an informative visualization of the transcription process, refer to the video "DrewBerry" available at wehi.tv, specifically at Timestamp: 3:22/7:19, which demonstrates gene start.
Page 9: Concurrent Transcription
Details About Transcription:
The transcription of a gene often transpires concurrently with the synthesis of multiple RNA molecules.
A fascinating example is the "Christmas Tree" electron micrograph, which vividly illustrates simultaneous RNA synthesis happening at once along a DNA template.
Noteworthy contributors to this groundbreaking discovery include Oscar Miller Jr, Barbara Hamkalo, and Charles Thomas, who conducted their studies in 1970.
Page 10: Template Strands
Template Strand Utilization:
One specific DNA strand acts as the template for the production of RNA molecules.
The nature of the template strand can vary: if the top strand serves as the template, the sequence of the bottom strand will match the RNA product and is referred to as the coding strand.
Page 11: Transcription Units
Definition of Transcription Unit:
A transcription unit is defined as a segment of DNA that encodes for an RNA molecule, inclusive of surrounding sequences necessary for transcription.
Adjacent Sequence Terms:
Upstream: Refers to the segment preceding the transcription start site.
Downstream: Indicates the sequences that follow the start of transcription.
Additional elements that define transcription units include promoter sequences and terminators, along with the coding strand necessary for the transcription process.
Page 12: Transcription Strand Utilization
Question on Strand Utilization:
Is it possible for both DNA strands to be used for RNA synthesis?Answers:
Yes, both strands can be transcribed under certain conditions.
No, typically only one strand is chosen as the transcriptional template.
Randomly, there exists a 50% probability that either strand could act as a template.
Page 13: Electron Micrograph Observations
What Does This Electron Micrograph Show?
This observation demonstrates a rapid succession of RNA transcripts being generated from a DNA template.
It emphasizes that DNA replication proceeds at an impressive pace, particularly during logarithmic growth phases observed in bacterial cells.
Additionally, the study analyzes the efficiency of CRISPR enzymes targeting specific DNA sequences.
Page 14: Promoters in Transcription Initiation
Initiation Mechanism:
The process of transcription initiation begins at specific sites known as promoters, which are unique DNA sequences that facilitate the binding of the RNA polymerase holoenzyme.
Bacterial promoters are characteristically located at the -10 and -35 regions, with the +1 position signifying the transcriptional start point.
The binding to the DNA is both specific and sequence-dependent; for example, the -10 consensus sequence frequently comprises TATAAT.
Page 15: Eukaryotic Promoters
Eukaryotes vs. Bacteria:
Eukaryotic organisms display a variety of promoters tailored to different RNA polymerases responsible for generating diverse RNA types.
In eukaryotic systems, promoters are more intricate, often concentrated around the -25 TATA box, which features a consensus sequence of TATAAA, critical for the initiation of transcription across multiple genes.
Page 16: Mutation Effects on Promoter Sequences
Consequence of Promoter Shift:
Shifting promoter regions upstream or downstream will significantly alter the starting point of transcription, affecting gene expression by the number of bases shifted.
Mutations occurring within the TATAAT promoter can severely diminish transcription efficiency due to alteration in the polymerase binding affinity.
Page 17: Transcription Outcomes with Promoter Shifts
Effect of Engineered Gene Changes:
If a promoter were to be shifted 10 bases upstream, it would likely cause the associated transcription to correspondingly change, with potential implications on gene expression levels.
This raises discussions on various hypothetical outcomes, such as whether transcription continues at the same original location or shifts as predicted.
Page 18: RNA Polymerase Specificity
RNA Polymerase Functionality:
A crucial examination of RNA polymerase reveals that not all RNA types produced in eukaryotes (namely rRNA, mRNA, and tRNA) are synthesized by RNA polymerase II.
This specificity underscores the complex regulatory mechanisms governing gene expression in eukaryotic cells.
Page 19: RNA Polymerase Recruitment
Role of Holoenzyme in E. coli:
In E. coli, RNA polymerase operates as part of a holoenzyme system that covers regions from -50 to +20 relative to the transcription start site.
Within bacterial systems, the sigma factor plays a pivotal role in binding the holoenzyme to the promoter sequence and unwinding the DNA at necessary locations, a crucial step to initiate transcription effectively.
Page 20: Eukaryotic Transcription Complex
Eukaryotic Transcription Complexity:
Eukaryotic transcription complexes are notably larger when compared to their bacterial counterparts, often containing approximately 50 subunits.
The TATA-binding protein (TBP) functions similarly to the sigma factor by forming linkages to the DNA and is essential for the stability of the initiation complex.
Initial transcription phases often face operational stalls termed abortive initiation, but these processes typically transition into promoter escape, progressing into the elongation phase.
The speed at which transcription occurs is approximately 40 bases per second, and the error rate averages around 1 in every 10,000 bases synthesized.
Page 21: Functionality of RNA Polymerase
Responsibilities of RNA Polymerase:
RNA polymerase unwinds the incoming DNA, establishing a short transcription bubble of approximately 18 nucleotides in length.
The enzyme features a funnel structure that accommodates the entry of ribonucleotide triphosphates (rNTPs) and directs the exit of synthesized mRNA.
Furthermore, RNA polymerase also plays a crucial dual role in both helicase and polymerase activities, facilitating the translocation process essential for RNA synthesis.
Page 22: Mushroom Toxicity
Discussion of Toxic Mushrooms:
Among the most perilous mushrooms found in the Northwest Region is the Death Cap (Amanita phalloides), which accounts for an alarming 90-95% of all fatal mushroom poisonings, primarily due to its severe liver failure effects.
The toxic effects of the Death Cap mushroom display a delayed onset, typically manifesting around 12-24 hours post-ingestion, complicating diagnosis and treatment protocols.
Another significant contributor to mushroom-related fatalities is The Destroying Angel (Amanita ocreata), noted for its similar lethal properties.
Page 23: Mechanism of Mushroom Toxicity
-amanitin and Its Effects:
-amanitin is a cyclic peptide that binds selectively to RNA polymerase II, subsequently obstructing transcription processes.
This binding occurs away from the active site of the enzyme, thus affecting the translocation step rather than directly interfering with the nucleic acid synthesis process.
Page 24: -amanitin Effects on Transcription
-amanitin's Mechanism:
The binding of -amanitin at promoter sites hinders the assembly of the basal transcription machinery, consequently obstructing the transcription initiation process.
It also prevents the access of ribonucleotide triphosphates (rNTPs) to the polymerase enzyme, effectively halting RNA synthesis.
Specifically, -amanitin attaches to RNA polymerase II and significantly impedes the translocation speed, markedly reducing the overall transcription rate.
Page 25: Summary of -amanitin's Actions
Overall Impact of -amanitin:
The inhibitory effects of -amanitin lead to a complete cessation of transcription activity, underscoring the compound's potent toxicity and its specific mechanism of action against RNA polymerase II, vital for synthesizing mRNA in eukaryotic cells.
Study Questions from Lecture 40 (12/2/2024)
Fat Cell Memory: Fat cells exhibit a form of "epigenetic memory," which may explain why it's easy to regain weight after losing it.
Types of RNA Produced: The two types of RNA produced through transcription that are important for translation are transfer RNA (tRNA) and ribosomal RNA (rRNA). Additionally, not all RNA is involved in translation; there are other types like microRNA (miRNA) and non-coding RNA (ncRNA). RNA does play a role in CRISPR technologies.
Christmas Tree Electron Micrographs: a. The DNA is represented by the template strands.b. The RNA is depicted as the elongated strands emerging from the DNA.c. The RNA polymerase enzymes move in the direction of transcription, typically 5' to 3'.d. The figure illustrates that multiple RNA polymerases can be transcribing concurrently on the same DNA strand, indicating that transcription can occur simultaneously rather than one polymerase at a time.
True/False Statements about Transcription vs DNA Replication: a. False: The source of energy for transcription comes from the triphosphate version of each ribonucleotide, similar to DNA replication.b. False: Both DNA replication and RNA synthesis proceed from 5' to 3'.c. True: Only one strand of DNA is used as a template in both processes.d. True: DNA replication requires primers, while transcription does not.e. True: Both processes occur in cells, but at different phases and times.f. True: Transcription occurs in specific regions while DNA synthesis involves the entire genome.
Transcription Unit: A transcription unit refers specifically to the segment of DNA that includes the sequences needed for transcription, while the region transcribed into RNA is part of this unit but does not encompass all surrounding elements that might be included in the transcription unit.
Promoter Functions: A promoter is a specific DNA sequence that facilitates the binding of RNA polymerase to initiate transcription. a. Yes, moving the promoter would change where transcription begins.b. In bacteria, the sigma factor binds at the promoter; in eukaryotes, the TATA-binding protein acts as the initiator.c. A consensus sequence is a sequence of DNA that is commonly found across different genes, indicating a regular pattern that is favored for transcription initiation.
Bacterial Holoenzyme Structure: The three arrows point to the transcription bubble, sigma factor, and promoter respectively. The sigma factor assists in binding the holoenzyme at the promoter and unwinding the DNA.
RNA Polymerase II Functionality: Calling RNA polymerase II both a polymerase and helicase means it both synthesizes RNA and unwinds the DNA helix during transcription.
α-amanitin Effects: α-amanitin is a cyclic peptide derived from certain mushrooms (e.g., Death Cap). It selectively binds to RNA polymerase II, inhibiting transcription without interfering at the active site but affecting the translocation process. The interaction occurs away from the active site where rNTPs enter.
Additional Notes:
α-amanitin is highly toxic and can lead to severe consequences including liver failure. Fly larvae might not be affected due to variations in their biochemical processes or potential mutations.
The liver is notably affected due to its role in metabolizing the toxin, resulting in disrupted protein synthesis and cell death, particularly impacting metabolically active tissues.
Questions:
What type of “memory” do fat cells appear to have that likely relates to why it is so easy to gain weight back once one has been overweight, but lost weight?
We often think of mRNA as the “product of transcription”. But actually, that’s a little misleading. All RNA is made through transcription. And yes, mRNA is the “transcript” that carries the genetic code from DNA to protein. What two kinds of RNA are produced by transcription that are really important for carrying out translation? Are there other types of RNA that are neither mRNA nor involved in translation? Does RNA play a role in CRISPR?
This depicts the “Christmas tree” electron micrographs of transcription.a. Where is the DNA?b. Where is the RNA?c. Which direction are the RNA polymerase enzymes moving?d. How does the figure illustrate address the question of whether transcription occurs one polymerase at a time vs being conducted concurrently?
RNA transcription has a lot of similarities to DNA replication. Here are some T/F statements to see if you can identify what is the same and what is different. If the statement is false, correct it.a. T/F: The source of energy for transcription (ATP) is fundamentally different than it is for DNA replication (where the energy comes from triphosopate version of each nucleotide).b. T/F: While DNA replication goes 5’ to 3’, RNA synthesis is just the opposite: 3’ to 5’.c. T/F: In both transcription and DNA replication, only one strand of the DNA—the template strand—is used.d. T/F: DNA replication involves primers that are later replaced, while transcription does not.e. T/F: Both DNA synthesis and transcription are ongoing processes in most cells for most of their existence.f. T/G: While DNA synthesis involves the whole genome, transcription is done in small specific regions of the genome.
How is a transcription unit different than the region of DNA that is transcribed into RNA (or are they the same thing)?
What is a promoter? That is, what occurs at a promoter?a. If I move (via genetic engineering) the promoter up- or down-stream by say 15 base pairs, will I change where transcription begins?b. In bacteria, what binds at the promoter to initiate the assembling of the holoenzyme? What about in eukaryotes—what is the name of this iniator protein?c. What is meant by a “consensus sequence?”
Here is a model (based on X-ray crystallography) of a bacteria holoenzyme that is initiating transcription. What do the three arrows point to? (Here are the three answers I’m looking for: transcription bubble, sigma factor, promoter).a. What does the sigma factor do?(If you want to spin this molecule around to get a better feel for its geometry, click here: https://www.rcsb.org/3d-view/6GH5)
Here is RNA polymerase II from eukaryotes. What do I mean when I say it’s both a polymerase and a helicase?
What is ⍺-amanitin? Where does it come from? What enzyme does it affect and how? Can you put an arrow to indicate where on the enzyme it binds? (Is it at the active site where base pairing occurs? Is in the pore where rNTPs enter?) Aside for those interested in aminita poisoning. This won’t be on the exam. Here is ⍺-amanitin (left) and a close up of ⍺-amanitin bound to RNA polymerase II (right). The arrow in the right panel points to a critical interaction between a hydroxyproline of the ⍺-amanitin compound and a charged glutamic acid at reside (site) 822 of RNA polymerase II. Glutamic acid is negatively charged. It’s also interesting to note that hydroxyproline (arrow in the left panel) is not one of the standard amino acids. So, the mushroom makes a chemical modification by adding a hydroxyl (OH) group. If chemists synthesize the ⍺-amanitin molecule with a proline instead of hydroxyproline, the toxicity of the molecule drops a whopping 20,000-fold! Presumably, this is because that interaction the arrow in the right panel points to doesn’t happen nearly as strongly—and thus translocation of the RNA Polymerase II enzyme is not inhibited. I have wondered if there are people (or animals) with mutations at site 822 and whether this would affects their susceptibility to ⍺-amanitin. When you find these mushrooms in the woods, you often find fly larvae have eaten them. I presume ⍺-amanitin doesn’t affect them; why? Today I noted that amanita poisoning is usually via liver failure. In this paper I found, it says “the liver is the principal organ affected, as it is the first organ encountered after absorption in the gastrointestinal tract.” This passage from the paper is also relevant: “Amanitins directly interact with the enzyme RNA polymerase II in eucaryotic cells and inhibit the transcription, causing a progressive decrease in mRNA, deficient protein synthesis, and cell death. For this reason, metabolically active tissues dependent on high rates of protein synthesis, such as the cells of the gastrointestinal tract, hepatocytes, and the proximal convoluted tubules of kidney, are disproportionately affected.
Additional Notes:
α-amanitin is highly toxic and can lead to severe consequences including liver failure. Fly larvae might not be affected due to variations in their biochemical processes or potential mutations.
The liver is notably affected due to its role in metabolizing the toxin, resulting in disrupted protein synthesis and cell death, particularly impacting metabolically active tissues.
Questions:
What type of “memory” do fat cells appear to have that likely relates to why it is so easy to gain weight back once one has been overweight, but lost weight?
We often think of mRNA as the “product of transcription”. But actually, that’s a little misleading. All RNA is made through transcription. And yes, mRNA is the “transcript” that carries the genetic code from DNA to protein. What two kinds of RNA are produced by transcription that are really important for carrying out translation? Are there other types of RNA that are neither mRNA nor involved in translation? Does RNA play a role in CRISPR?
This depicts the “Christmas tree” electron micrographs of transcription.a. Where is the DNA?b. Where is the RNA?c. Which direction are the RNA polymerase enzymes moving?d. How does the figure illustrate address the question of whether transcription occurs one polymerase at a time vs being conducted concurrently?
RNA transcription has a lot of similarities to DNA replication. Here are some T/F statements to see if you can identify what is the same and what is different. If the statement is false, correct it.a. T/F: The source of energy for transcription (ATP) is fundamentally different than it is for DNA replication (where the energy comes from triphosopate version of each nucleotide).b. T/F: While DNA replication goes 5’ to 3’, RNA synthesis is just the opposite: 3’ to 5’.c. T/F: In both transcription and DNA replication, only one strand of the DNA—the template strand—is used.d. T/F: DNA replication involves primers that are later replaced, while transcription does not.e. T/F: Both DNA synthesis and transcription are ongoing processes in most cells for most of their existence.f. T/G: While DNA synthesis involves the whole genome, transcription is done in small specific regions of the genome.
How is a transcription unit different than the region of DNA that is transcribed into RNA (or are they the same thing)?
What is a promoter? That is, what occurs at a promoter?a. If I move (via genetic engineering) the promoter up- or down-stream by say 15 base pairs, will I change where transcription begins?b. In bacteria, what binds at the promoter to initiate the assembling of the holoenzyme? What about in eukaryotes—what is the name of this iniator protein?c. What is meant by a “consensus sequence?”
Here is a model (based on X-ray crystallography) of a bacteria holoenzyme that is initiating transcription. What do the three arrows point to? (Here are the three answers I’m looking for: transcription bubble, sigma factor, promoter).a. What does the sigma factor do?(If you want to spin this molecule around to get a better feel for its geometry, click here: https://www.rcsb.org/3d-view/6GH5)
Here is RNA polymerase II from eukaryotes. What do I mean when I say it’s both a polymerase and a helicase?
What is ⍺-amanitin? Where does it come from? What enzyme does it affect and how? Can you put an arrow to indicate where on the enzyme it binds? (Is it at the active site where base pairing occurs? Is in the pore where rNTPs enter?) Aside for those interested in aminita poisoning. This won’t be on the exam. Here is ⍺-amanitin (left) and a close up of ⍺-amanitin bound to RNA polymerase II (right). The arrow in the right panel points to a critical interaction between a hydroxyproline of the ⍺-amanitin compound and a charged glutamic acid at reside (site) 822 of RNA polymerase II. Glutamic acid is negatively charged. It’s also interesting to note that hydroxyproline (arrow in the left panel) is not one of the standard amino acids. So, the mushroom makes a chemical modification by adding a hydroxyl (OH) group. If chemists synthesize the ⍺-amanitin molecule with a proline instead of hydroxyproline, the toxicity of the molecule drops a whopping 20,000-fold! Presumably, this is because that interaction the arrow in the right panel points to doesn’t happen nearly as strongly—and thus translocation of the RNA Polymerase II enzyme is not inhibited. I have wondered if there are people (or animals) with mutations at site 822 and whether this would affects their susceptibility to ⍺-amanitin. When you find these mushrooms in the woods, you often find fly larvae have eaten them. I presume ⍺-amanitin doesn’t affect them; why? Today I noted that amanita poisoning is usually via liver failure. In this paper I found, it says “the liver is the principal organ affected, as it is the first organ encountered after absorption in the gastrointestinal tract.” This passage from the paper is also relevant: “Amanitins directly interact with the enzyme RNA polymerase II in eucaryotic cells and inhibit the transcription, causing a progressive decrease in mRNA, deficient protein synthesis, and cell death. For this reason, metabolically active tissues dependent on high rates of protein synthesis, such as the cells of the gastrointestinal tract, hepatocytes, and the proximal convoluted tubules of kidney, are disproportionately affected.