Eukaryotic Transcription And Translation Flashcards
Compare a mitochondrial genome to a chloroplast genome
- Mitochondria and chloroplasts divide independently of cell division
- Numbers of mitochondria between 50-millions compared to 0-60 chloroplasts
- Maternal inheritance in mitochondria
- Chloroplast has a much larger genome
How did organelles come to have DNA
- Simple prokaryotes (proteobacterium) were endocytosed into a larger anaerobic cell
- Proteobacterium + anaerobic cell have a symbiotic relationship due to the ability of the prokaryote to produce energy via aerobic respiration
- Cyanobacteria had the ability to photosynthesise and respire
Describe the types, structure and function of histones
- Five types, H1, H2A, H2B, H3 and H4 histones
- 4 pairs/octamer of histone proteins plus H1 histone that form the nucleosome
- Wrap DNA around them, condensing them and making them inaccessible, but capable of unwrapping when DNA is required
What is chromatin
- The combination of DNA and the proteins it is associated with
Give an example of non-histone protein function
- Metaphase chromosome maintains its shape thanks to non-histone protein scaffold
What is the visible manifestation when chromatin is loosened
- Puff regions show uncoiled DNA and correspond to the high gene activity
Contrast heterochromatin and euchromatin
- Heterochromatin is strongly associated with histones and highly condensed, whereas euchromatin is uncoiled and less condensed
2 heterochromatin is therefore inaccessible and genetically inactive, whereas euchromatin is accessible and can be transcribed
- Heterochromatin found in eukaryotes, whereas euchromatin found in both prokaryotes and eukaryotes
Describe and explain the effects of histone modification (one tail per histone = 8 tails per nucleosome)
- Lysine in histone tails can be acetylated and methylated
-Acetylation results in heterochromatin shifting to euchromatin
-Methylation results in tighter histone association and thus silences gene expression - Serine in histone tails can be phosphorylated
-phosphatation prepares the chromatin for mitosis/meiosis (extreme condensation?) - Ubiquitination marks damaged or old proteins for destruction and/or recycling
Explain the role of the chromatin remodelling complex in transcription
- Chromatin remodelling complex uses ATP to bind to DNA, repositioning nucleosomes so that a transcription factor binding site is revealed
- This initiates the assembly of the transcriptional apparatus
Why is eukaryotic gene expression so variable
- Complex organisms must respond to a wide range of changing stimuli
- Multicellular organisms have cells that fulfill different purposes and need different proteins
- Upregulation, downregulation and silencing are thus essential for multicellularity
Give two examples of eukaryotic genes switching on and off
- The photo activation of photosynthetic genes
- Hormone-receptor interaction leads to a receptor signal that interacts with regulatory region of a hormone-responsive gene
Differences in transcription between eukaryotes and prokaryotes
- Transcription spatially separated from translation by the nuclear envelope
- Different RNA polymerases are used in eukaryotes
RNA pol II most active in eukaryotic transcription - Splicing, addition of 5’ cap and poly-A-tail occurs in eukaryotes (transcript processing)
- More components in the transcriptional assembly in eukaryotes
- Heterochromatin must be converted to euchromatin in order for transcription to occur
- There is post-transcriptional control -
Describe the three RNA pol forms
- RNA pol I in the nucleolus and synthesises rRNA
-not sensitive to a-amanitin - RNA pol II in the nucleoplasm and synthesises hnRNA (protein coding genes)
-high sensitivity to a-Amanitin toxicity - RNA pol III in the nucleoplasm and synthesises tRNA and small RNAs (snRNAs)
-medium sensitivity to a-amanitin
Purpose of alternative splicing
To produce a variety of different mature mRNAs from the same pre-mRNA sequence
If certain splice factors are present, exons may be spliced out or spliced constitutively (kept), different 3’ or 5’ splice sites, introns kept
What components make up the Pre-initiation complex
- General transcription factors (TFIID/B/F/G/H) and RNA polymerase II bind to the TÂTA box, a region called the core promoter region of the gene.
Functions of the general transcription factors GTFs
- Position RNA poll II at the TATA box
- Allow TATA-binding protein to then bend and separate DNA strands to initiate transcription
- The equivalent of the sigma factors in prokaryotes
Which TFs are utilised as a response to a stress stimulus that can also achieve co-ordinate control
- Specific transcription factors bind to cis element sequences in the regulatory promoter region
- This usually activates general transcription factors to bind to the core promoter (TATA box) initiating transcription
- A specific transcription factor can bind to all cis sequences present independent on how close genes are to each other, thus one TF can regulate the expression of multiple genes at once.
- Triggered usually due to cell draught, stress or light etc.
How are transcription factors’ transcription regulated
- Upon stresses or stimuli, the genes for the transcription factors can regulate the transcription OR translation of the TF gene
- Usually the stability of the protein is stabilised by the draught/cold OR binding is facilitated more easily which allows them to fulfill their usual activity
Sugggest a reason why organisms with similar genomes can have such similar phenotypes
- Different patterns of TF expression and therefore levels of gene expression is the main reason phenotype between humans and chimps are so different despite sharing 99% genome similarity.
What are all the components required for transvription
- TFs
- Poll II
- GTFs
- Co-activator - identified to be the ‘Mediator co-activator complex’
Structure + Function of the Mediator
- Brings TFs and RNA pol II together to more efficiently form the pre-initiation complex
- Mediator is a multi-protein complex composed of a head attached to RNA poll II, middle and tail which interacts with transcription factors, together with a regulatory kinase attached.
Give an example of a mutation in the mediator complex which lead to deleterious impact
- Anti-freeze gene was not as highly expressed as the wild-type
- Defective mediator tail subunit
- Little effective communication between the TFs and the RNA Pol II
- The antifreeze protein is plants was therefore lower and caused the plant respond negatively in the presence of cold
Give an example of de-acetylation in flowers
- Acetyl groups keep the FLC gene open for transcription
- Transcribed and translated into a regulatory protein which represses flowering
- Plants have a FLD gene which encodes a deacetylase enzyme which removes acetyl groups meaning FLC is not expressed, facilitating flowering
Describe the structure+formation+general function of small RNAs
- Double-stranded non-coding RNA
- Formed via the DICER enzyme which chops a long dsRNA into pieces
- General function is to silence/reduce gene expression at the post-transcriptional level
Explain the formation and function of Small interfering RNAs (siRNA)
- Formed via the cleavage of dsRNA by DICER
- An siRNA combines with proteins to form RISC (RNA induced silencing complex)
- Since siRNA is complementary to its own genes, this gene is targeted by RISC
- RISC cleaves mRNA leading to degradation
- SLICER catalyses cleavage triggered by siRNAs
- Can ALSO attach to complementary mRNA sequence inducing histone methylation inhibiting transcription
Explain the formation+function of micro RNAs (miRNAs)
- Formed via the cleavage of single stranded RNA hairpins by DICER —> miRNAs
- MiRNAs + proteins = RISC
- The binding to mRNA is not perfect so translation is inhibited, but mRNA not cleaved
Which mechanism leads to miRNAs degrading mRNA (miRNAs usually inhibit translation)
- Some miRNAs complementary to an AU-rich region of the 3’ UTR end of mRNA activate DICER and can lead to mRNA degradation
Other than gene inhibition what other roles does miRNAs fullfil
- MiRNAs secreted via exocytosis outside the cell
- Mediate cell-cell communication
- De-regulation of miRNAs linked to cancer
Describe the composition of the 70s and 80s ribosomes and explain what 70s or 80s mean
- 70s is found in bacteria and has smaller subunits made of fewer rRNAs and proteins than 80s found under eukaryotes
- 70s stands for the distance that the ribosomes sink when centrifuged in sucrose. Bigger = move more so 80s sinks more than 70s
Describe the structure and role of tRNA
TRNA provides the complementary amino acid to the mRNA codon
Each amino acid has a specific tRNA
Amino acid bonds to the acceptor arm
The variable loop and tRNA arms help to differentiate between different tRNAs
Describe the structure and roles of aminoacyl tRNA synthetases
- Aminoacyl tRNA synthetase joins amino acid -AMP to the tRNA
- There is a specific aminoacyl synthetase for each amino acid
- AMP attachment required for enzyme to work
- enzyme can identify incorrect amino acids and move them to the editing site where a-acid is hydrolysed and removed
Explain the process of translation - Initiation
- First codon on mRNA always AUG, methionine
- Special initiator tRNA for start methionine that is different for other methionines
- Bacteria one called N-formylmethionine
- Small subunit binds to ribosome binding site with IF-3 to stop large subunit binding
- Special initiator tRNA binds
- Large subunit added
- Subunit addition catalysed by initiation factors
- Prokaryotes have a shine-dalgarno sequence which rRNA is complementary to helping ribosome bind to the mRNA
- In eukaryotes the 5’ cap is recognised by IFs, polyA tail used for small subunit binding, and the Kozak sequence at the start codon is identified by ribosome
Explain the process of elongation and translocation
- Aminoacyl-tRNA bonds to the P site
- New aminoacyl-tRNA bonds to A site
- EF-1A hydrolyses GTP to GDP which gives energy to make peptide bond
-Peptidyl transferase is the enzyme that catalyses peptide formation - EF-1B removes GDP and adds GTP back onto EF-1A
- Deacylated tRNA falls off due to EF-2A GTP hydrolysis during translocation
Explain the process of termination
- RF-1 and RF-2 (releasing factor) occupies the A site
- Amino acid in P site gains water instead of a peptide bond
- RRF takes apart the subunits
Describe the structure of+function of rRNA
- RRNA catalyses peptide bond synthesis
- Located on the small and large subunits
- RRNA is folded such that G-U pairings can occur (weird folding)
How does codon degeneracy physically happen
- The third position in the anticodon undergoes relaxed binding
- This is due to the curved shape of the anticodon that allows the third position to be the ‘wrong base’
- Called wobble-pairing
- Hence why most amino acids will corresponding sequences with the first two letters the same but the last letter different
Explain the mechanism that deals with problematic mRNAs stuck in the ribosome in prokaryotes
- Ribosome stalls when there is no stop codon
- TmRNA is charged with Ala binding to the A site
- Translocation can continue adding new amino acids ~10 alanines
- TmRNA contains a stop codon
- RF-1 and RF-3 together with GTP hydrolysis leads to termination
- The alanine peptide is recognised and labelled for degradation
What are the reasons for why the genetic code is in triplets
How was the triplet code proven
- With 20 amino acids a doublet based code, only 16 combinations of sequences, and 3 would give 64 combos
- By adding one nucleotide, there was a frame shift but removing one put the sequence back into frame
Adding two nucleotides muck it up
Adding three was very similar to wild type
Explain nirenberg’s first experiment to examine which rna specified an amino acid
- Polynucleotide phosphorylase used to make a single base polymer
- Created a cell-free extract containing ribosomes and tRNA and an enzyme to destroy any other dna
- One sample had radiolabelled amino acid
- The radiolabelled protein would be made from a polypeptide made from only one amino acid
- Made random copolymers = took a while with lots of uncertainty
- Another scientist, Khorana, provided exact nucleotide sequences which could then be tested with each amino acid
- tRNAs were used - code of mRNA was known, one a-acid radiolabelled so anticodon could be figured out and corresponded to the a-acid
Describe the order of events during transcript processing
- Intron excision and exon ligation
- splicing carried out by the spliceosome - 5’ Cap added and poly A tail added by polyA polymerase
