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?
Where than the genes being transcribed be found on 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 is this made possible?
What does this method allow to be controlled?
- 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 do promoters contain?
What does this allow?
What is the promoter for RNA polymerase 2?
Why does it have this structure?
Where is it found?
- Promoters contain specific DNA sequences, such as response elements, that provide a secure initial binding site for RNA polymerase and for proteins called transcription factors, which recruit and bind RNA polymerase
- The promoter for RNA polymerase 2 is known as the TATA box, as it contains a TATA rich sequence
- Since AT base pairs contain 2 hydrogen bonds instead of the 3 found in GC pairs, less energy will be required to separate the 2 strands of DNA at this point
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?
- Transcription factors are proteins that control the rate of transcription by binding to a specific DNA sequence
- The function of transcription factors is to regulate (turn on and off) genes to make sure they are expressed in the right cell at the right time
- They also recruit and bind RNA polymerase to promoter regions
- The transcription factor associated with the TATA box promoter is TBP (TATA box binding protein)
- This is found in the core promoter region (0 – 50 bases before transcription initiation site)
- Gene specific transcription factors can be found in the proximal promoter region (50 – 200 bases from initiation site)
- These TFs can allow more precise regulation of when genes are expressed, and contribute to the overall initiation of transcription
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?
- Enhancers are short regions of DNA
- Functions of enhancers:
- Enhancers can be bound by transcription factors to increase likelihood that transcription of a particular gene will occur.
- They stabilised the transcription machinery assembly by protein-protein interactions
- Enhancers help the transcription complex gain enough unwinding of the DNA, and ensures circumstances are right for RNA polymerase to initiate transcription
- Enhancers are position and orientation independent (unlike promoters), and will work no matter how far it is from the gene
- Enhancers can be quite a distance back from the initiation site of transcription
- Enhancers loop round and can be bound by transcription factors, allowing them to work from a distance?
- Enhancers are opposed by silencers
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?
- We can develop artificial repeater genes and insert sequences if we think they may be part of the promoter or enhancer
- We can generate this artificial gene, and test it in a cell culture system, to see if there are changes in gene regulation
- The reporter gene can fluoresce under a microscope when there has been transcription and translation of the gene (in this example, green)
- There may be a region in front of the reporter gene that can only bind transcription factors under certain circumstances (in this example, hypoxia, which displays the genes ability to respond to low oxygen level)
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 around 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
