28-10-21 - Introduction to Molecular Biology 3 Flashcards
What are 3 differences in the secondary structure of DNA and RNA?
- RNA is not consistently folded in a base paired manner, compared to DNA’s double helix structure
- RNA contains a lot of pseudoknots and complex structures
- RNA secondary structure is a lot less stable than DNA’s secondary structure.
What is DNAs only function?
What are the 5 functions of RNA?
• DNA’s exclusive function is storage of genetic information
- The 5 functions of RNA
- Genetic messages – messenger RNA (Mrna)
- Guide RNAs, such as IN telomerase
- Adaptor RNA – transfer RNA (Trna)
- Structural RNA – Xist (non-coding RNA on x chromosome of placental mammals)
- Catalyst – ribosome (Rrna) and spliceosome (snRNA)
What unwinds DNA for transcription?
What are the 2 strands referred to as?
What does RNA polymerase need to become active?
What direction does it work in?
Are all genes that are transcribed found on same strands of chromosomes?
How does transcription compare to DNA replication in terms of accuracy?
- RNA polymerase and access proteins in wind the DNA by breaking the hydrogen bonds
- One strand of DNA will be used as the template strand
- The other strand is known as the coding strand, as the RNA will have the same base sequence as this strand
- RNA polymerase, unlike DNA polymerase, does not require primers to work but does require accessory proteins in order to become active.
- RNA polymerase also works in the 5’ to 3’ direction
- It is important to note that perhaps not all the genes that are transcribed are found on the same strands of chromosomes.
- They can potentially be interspersed
- RNA transcription is less accurate than DNA replication, as Mrna has a short life span, so errors are not as serious
- DNA riddled with errors can cause more mutations, which can have serious effects
How many copies are made in DNA replication compared to RNA transcription?
How do the RNA polymerases work?
what is controlled/turned on?
- In DNA replication, only 1 copy is generated
- In RNA transcription, multiple chains are generated in order to make the most efficient use of the DNA
- This is made possible by multiple RNA polymerases working simultaneously to generate many strands of RNA
- This is one way gene expression is controlled
- If the gene expression is turned on, multiple polymerases will move along that gene
How does the RNA polymerase know where to start on the DNA strand during transcription?
How does it know when to stop?
- There promotor is a sequence just before the transcription start site
- Proteins, such as accessory proteins, can be recruited to this area, which allows RNA polymerase to start transcribing at a set position
- There are also bases in the terminator area which tells RNA polymerase to stop transcription and fall off DNA
What are the 3 different types of RNA polymerases?
What types of RNA do they generate?
- RNA polymerase 1 – Rrna (ribosomal RNA)
- RNA polymerase 2 – Mrna (protein coding)
- RNA polymerase 3 – Trna (transfer RNA)
What are promoters and what do they contain?
What does this allow?
What is the promoter for RNA polymerase 2?
Why does it have this structure?
Where is it found?
Here’s a simpler explanation of this concept:
What is a Promoter?
- A promoter is a region of DNA that helps start the process of transcription (making RNA from DNA).
- It contains specific DNA sequences, called response elements, where RNA polymerase and transcription factors (helper proteins) bind. Together, they prepare RNA polymerase to begin copying the gene.
The TATA Box:
- For RNA polymerase II (the enzyme that makes RNA for most protein-coding genes), the promoter often includes a sequence called the TATA box.
- The TATA box is a stretch of DNA rich in the letters T (thymine) and A (adenine).
Why TATA is Special:
- T and A in DNA are connected by 2 hydrogen bonds, while G (guanine) and C (cytosine) are connected by 3 hydrogen bonds.
- Since TATA sequences are easier to pull apart than GC-rich sequences, RNA polymerase and transcription factors can separate the DNA strands more easily to start transcription.
Summary:
- The TATA box is like a weak spot in DNA that makes it easier for transcription to begin.
- It helps RNA polymerase II bind and separate the DNA strands where the gene starts.
What are transcription factors?
What are 2 functions of transcription factors?
What is the transcription factor associated with the TATA box promoter?
Where is this found?
What else can be found further back?
What is the function of these?
Here’s a simpler explanation of the role of transcription factors and promoters:
What Are Transcription Factors?
- Transcription factors (TFs) are proteins that help control which genes are turned on or off.
- They work by binding to specific DNA sequences, often near the start of a gene, and controlling the rate of transcription (copying DNA into RNA).
Where Do Transcription Factors Work?
1. Core Promoter Region (0–50 bases before the start of the gene):
- This is the main site where transcription begins.
- A key transcription factor here is TBP (TATA box binding protein), which binds to the TATA box (a DNA sequence in many promoters).
- TBP helps recruit RNA polymerase, the enzyme that copies DNA into RNA.
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Proximal Promoter Region (50–200 bases before the start of the gene):
- Gene-specific transcription factors bind here.
- These TFs provide more precise control over gene activity, ensuring the gene is only active in the right cell, at the right time, and under the right conditions.
What Do Transcription Factors Do?
1. Turn Genes On or Off: They regulate when genes are expressed (used to make proteins).
2. Recruit RNA Polymerase: They help RNA polymerase find the promoter so it can start transcription.
3. Fine-Tune Gene Expression: Gene-specific TFs in the proximal promoter make transcription more controlled and responsive to the cell’s needs.
Summary:
- Transcription factors bind to specific parts of DNA to control how and when genes are used.
- TBP in the core promoter helps RNA polymerase start transcription.
- Other TFs in the proximal promoter add extra control, making gene expression more precise.
What are housekeeping genes? Where are they expressed? What is an example of these?
- Housekeeping genes are typically genes that are required for the maintenance of basic cellular function, and are expressed in all cells of an organism under normal patho-physiological conditions
- Actin is required to maintain the cytoskeleton of the cell, so its expression will never change very much
What are enhancers?
What are 3 functions of enhancers?
How do they contrast from promoters?
Where are they in relation to the initiation site of transcription?
How do they work from here?
What opposes enhancers?
Certainly! Here’s a clearer organization of the information about enhancers:
Enhancers
Definition:
- Short regions of DNA that regulate gene transcription.
Functions:
1. Transcription Activation:
- Enhancers can be bound by transcription factors to increase the likelihood of transcription for a specific gene.
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Stabilization of Transcription Machinery:
- They stabilize the assembly of the transcription machinery through protein-protein interactions.
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Facilitation of DNA Unwinding:
- Enhancers assist the transcription complex in unwinding DNA, creating conditions favorable for RNA polymerase to initiate transcription.
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Position and Orientation Independence:
- Unlike promoters, enhancers are position and orientation independent, meaning they can function regardless of their distance from the gene they regulate.
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Distance Flexibility:
- Enhancers can be located far upstream or downstream from the transcription initiation site and can still exert their effects.
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DNA Looping Mechanism:
- Enhancers can loop around to interact with the transcription machinery, allowing them to work from a distance.
Opposition:
- Enhancers are countered by silencers, which inhibit transcription.
This format provides a clear overview of the roles and characteristics of enhancers in gene regulation.
What are the 2 domains of transcription factors?
What are they?
What are their functions?
- DNA binding domain
- Proteins folded in a particular way that aligns precisely with certain sequences of bases in the DNA
- This allows transcription factors to bind to DNA
- The activation domain
- The part of the transcription factor that can form a complex with other proteins
- This stimulates RNA polymerase and ensures the conditions are right to initiate transcription
What are the 2 ways chromatin is modified to allow RNA polymerase access?
- Histone-modified enzyme
* Chromatin remodelling complex remodels nucleosomes
What are the fast and slow intracellular responses to extracellular signal molecule binding to cell-surface receptors?
What do both together lead to?
- Fast response (less than a second to minutes)
- E.g hunger signal due to being close to eating
- When the extracellular signal molecule binds, this quickly initiates an intracellular pathway that causes altered protein function
- This can cause enzyme secretion via exocytosis e.g amylase released in the mouth to digest starch
- Slow response
- Enzymes secreted in the fast response now must be replaced
- There is slower intracellular signalling that tells the nucleus to replace the enzymes secreted
- This results in transcription taking place in the nucleus and altered protein synthesis in the cytoplasm to generate new enzymes
• Both fast and slow responses lead to altered cytoplasmic machinery, and altered cell behaviour
How can we determine what parts of DNA are responsible for binding transcription factors and contributing to gene regulation?
How can this be tested?
What might be present Infront of what we created?
Here’s a simplified explanation of what this means:
Testing Gene Regulation Using a Reporter Gene
Scientists can study how certain DNA sequences (like promoters or enhancers) affect gene regulation by using an artificial system. Here’s how it works:
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Create an Artificial Gene:
- Scientists build a piece of DNA that includes:
- A reporter gene: This is a gene that produces a detectable signal, such as fluorescence (e.g., glowing green under a microscope).
- A potential promoter or enhancer sequence: This is the DNA region being tested to see if it affects transcription (how RNA is made).
- Scientists build a piece of DNA that includes:
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Test It in Cells:
- The artificial gene is inserted into cells in a lab (called cell culture).
- If the promoter or enhancer is active, it will recruit transcription factors, allowing transcription and translation of the reporter gene.
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Detect the Reporter Signal:
- When the reporter gene is expressed, it produces a visible signal (e.g., glowing green fluorescence).
- This indicates that the tested promoter or enhancer is functional.
Specific Example: Hypoxia
- Scientists might test whether a DNA sequence responds to hypoxia (low oxygen levels).
- They attach the sequence to a reporter gene and place it in a cell culture under low-oxygen conditions.
- If the DNA sequence activates the gene in response to hypoxia, the cells will fluoresce, showing that this region can bind transcription factors related to low oxygen.
Why Is This Useful?
- It helps identify specific DNA regions that regulate genes under different conditions (e.g., low oxygen, stress, or hormones).
- It’s a powerful tool to study how genes are controlled and to understand their role in health and disease.
Summary:
Scientists create artificial DNA with a reporter gene and test it in cells to see if specific DNA sequences (like promoters or enhancers) regulate gene expression. For example, under low oxygen conditions (hypoxia), the glowing green signal shows that the tested sequence responds to this environment.
What are 6 different ways transcription can be activated?
What are some examples of proteins that use each method?
• There are many ways genes can be switched on and transcribed to produce RNA
1) Absence of transcription factor e.g homeoproteins
2) Protein phosphorylation – protein can’t bind DNA till phosphorylated e.g HTSF (heat shock transcription factor)
3) Protein dephosphorylation e.g AP1
4) Ligand binding – protein made in wrong compartment of the cell but can be moved to the right place in response to specific signals, like ligands (e.g transcription factors moving from cytoplasm to nucleus) e.g Steroids and receptors
5) Released from an inhibitor – Proteins in an inactive conformation due to binding an inhibitor. In response to signals (e.g inhibitor degrader) this can allow the protein to become active and move to a new location e.g NFkB
6) Change of binding partner – can change binding partners depending on the proportion of each partner within a cell e.g HLH
What is a condition caused by an absence of a transcription factor?
What transcription factor is this?
Why does it produce similar conditions when absent in different species?
- Aniridia Is the absence of an iris in the eyes
- It is cause in humans by the absence of transcription factor PAX6
- PAX6 is highly conserved in evolution, so absence of PAX6 (or equivalent) In other species will produce similar results
- is a working copy and – is a mutated copy
What are the 2 modifications of pre-messenger RNA?
What are the 3 reasons for these modifications?
- RNA capping
- RNA transcription usually starts with a G
- A methyl G cap is added to the start of the pre-messenger RNA at the 5’ end after RNA polymerase moves around 25 bases away
- Polyadenylation
- At the end of the pre-messenger RNA at the 3’ end, polyadenylation is added, which is a chain of repeating A bases 150-250 bases long
- The 3 reasons for these modifications:
- Protect from exonucleases, as RNA is unstable
- Aids export into cytoplasm from nucleus for translation to occur
- To identify the strand as mRNA, as opposed to trna, rrna or telomerase rna
What is the structure of DNA that codes for proteins in terms of coding and non-coding regions?
What does this mean for the Mrna produced form this DNA?
What must happen to this pre-mRNA?
What does this produce?
What is it used for?
How many bases for an amino acid?
How can this be spared in exons?
- The vast majority of genes that code for proteins contain regions of DNA which encode for proteins (exons), interspersed by regions which do not encode proteins (introns)
- Transcription of this DNA produces pre-mrna, which contains both the introns and exons
- These introns need to be spliced out in order to produce mature mRNA, which has an open reading frame that only contains exons
- This mature Mrna is then moved to the cytoplasm for translation
- 3 bases code for each amino acid, but each exon isn’t always divisible by 3.
What process is responsible for splicing?
What is it composed of?
What do these subunits contain?
What kind of reaction does RNA splicing take place through?
What are the 2 stages of splicing?
- Spliceosomes are responsible for RNA splicing
- Spliceosomes are complex multi-component proteins usually containing 25-30 proteins
- These proteins carry snRNA (small nuclear RNA), which recognise specific sequences in the pre-mRNA at the intron-exon junction, and sequences embedded within the intron
- Splicing takes place via esterification reactions
1) Branch site attacks intron-exon junction, which forms a free hydroxide group at the 3’ end of the exon and a lariat structure
2) This hydroxide group then attacks another intro-exon junction in order to release the lariat structure, which leaves a splices exon Mrna
How are exons recognised in the pre-mRNA by spliceosomes?
What does this contribute to?
- The snRNA in the subunit proteins of spliceosomes look for sequences in the intron-exon junction and around the branch site
- They then form a base pairing with these sequences
- This contributes to the accuracy of splicing.
How can splicing produce different proteins?
What does this produce?
What is a rule of this process?
- Alternative RNA splicing can cause exon skipping, meaning an exon is left out from the mature mRNA after splicing
- This results in the production of different mRNA transcripts, which produce different protein isoforms (protein variants) with different functions
- Exon order is always maintained.
What is Frasier syndrome characterized by?
What causes Frasier syndrome?
What exon is this associated with?
Where is it found?
What proteins does it code for?
What are the differences between these proteins?
What problem leads to Frasier syndrome?
What does this highlight?
- Frasier syndrome is characterized kidney disease, which involves a condition called focal segmental glomerulosclerosis (FSGS), where scar tissue forms in the kidney glomeruli.
- Frasier syndrome is caused due to consequences in a tumour suppressor gene
- A transcription factor contains a zinc finger structure in its DNA binding domain
- Exon 9 is located between fingers 3 and 4, is 3 amino acids long, and codes for 2 different proteins: +KTS and -KTS
- -KTS is a more solid structure, whereas +KTS is a structure that contains more gaps.
- The are both nuclear proteins, but -KTS is a transcription factor, and +KTS is a splicing factor
- Patients have mutations in the non-coding intron around exon 9, which results in less production of +KTS
- This highlights the importance of regulation of splicing, and how changes in non-coding regions can have serious effects on health.
What are the 5 ways in which different proteins can be made form the same gene?
1) Alternative promoters
2) Alternative splicing
3) Alternative 3’ ends - causes alternative transcription termination
4) RNA editing
5) Translational control
