M1 - Genomes and Evolution Flashcards
Draw an alpha glucose, beta glucose and the dynamic interchange.
Alpha glucose is about 36% abundant and OH is on the bottom. Beta glucose is about 64% abundant and OH is on the top.
Briefly describe carbohydrates.
Glycogen: multibranched alpha-glucose polysaccharide used for energy storage in animals, fungi and bacteria.
Cellulose: beta-glucose molecules are linked to form fibres that give structured cell walls in plants and algae.
Briefly describe lipids.
A water-insoluble biomolecule which is highly soluble in organic solvents like chloroform. For energy, we use fatty acids. For structure, we use membrane lipids and for signalling we use steroid hormones.
Briefly describe amino acids.
20 amino acids commonly found in proteins. Altering the R side chains alters the properties of an amino acid, the primary structure folds into the mature form of the protein. The polarity is read from N- to C- terminus.
Briefly describe nucleotides and nucleic acids.
Nucleotides are joined to form nucleic acids, nucleic acids store genetic information.
Describe ‘The Great Chain Of Being’ and who came up with the kingdom of animals?
La Scala Naturae. A medieval view of the divisions of life as an ascending stairway: lower forms to higher forms. The higher the being is in the chain, the more attributes it has including all the attributes of the beings below it. Linnaeus’ came up with the idea of the Kingdom of Animals.
Describe Charles Darwin and diverging lineages. Who else did something similar to this?
In 1837, Charles Darwin made the “Tree of Life” Sketch. This showed diverging lineages. Edward Hitchcock (1840) also did it.
Who redefined evolution after Charles Darwin and what was their conclusion?
Theodosius Dobzhansky in 1937 redefined evolution to be a change in allele frequency in a gene pool.
Describe what cetaceans are.
They are whales, dolphins and porpoises.
Describe homologous traits and their relation to shared ancestry.
Homologous traits are useful for constructing phylogenetic trees. Homology: any similarities between traits because of shared ancestry. These traits are called derived traits or apomorphies.
What is an involucrum?
Synapomorphy among cetaceans.
What are synapomorphies?
Derived form of traits shared by two or more taxa.
What can a distinctly shaped astragalus (ankle bone) show?
Synapomorphy among artiodactyls.
Describe phylogenetic terminology: Terminals, nodes, roots, cladistics.
Terminals: The end of the phylogenetic tree.
Nodes: Branch point that represents a common ancestor.
Roots: Represents the ancestral population.
Cladistics: classification where organisms are grouped by most recent common ancestry.
Are nodes read at tips or ends and why?
Branches can rotate around nodes which means the label order changes but relationship doesn’t.
Describe what a monophyly and paraphyly is.
Monophylyl: A clade comprises an ancestor and all its descendants.
Paraphyly: An ancestor and some but not all its descendants.
What is an autapomorphy?
It’s a self-derived character. A derived trait that is unique to a given taxon.
Describe the similarities between eukaryotes and archaea.
A comparison of genomes and protein functional studies suggested similarities between eukaryotes and the TACK archaea in:
- Proteins involved in cytokinesis (cell division)
- Cell shape determination
- Protein recycling
- Membrane remodelling (cellular compartmentalisation)
What is microevolution?
Changes in allele frequencies in a population of a species over time. 3 main mechanisms:
- natural selection
- genetic drift
- gene flow
What is macroevolution?
Change at or above the level of the species.
Describe Darwin’s hypothesis (1859).
For any particular trait, individuals within a species are variable.
At least some of the variation is heritable.
Reproduction is not random but is selected by nature, individuals that reproduce the most are those with the most favourable variations.
Describe who tested Darwin’s Hypothesis.
Rosemary and Peter Grant (1976-1978). They proposed that parents of birds with small beaks tend to have offspring with shallow beaks. Parents with deep beaks tend to have offspring with deep beaks. Hence there’s a large genetic component to determination of beak depth.
Describe the way that natural selection is linked to the Chernobyl nuclear disaster. What was the selective pressure for this?
This disaster happened in 1986 and natural selection has selected for melatonic (Dark) Eastern tree frogs in pools inside the Chernobyl exclusion zone and paler frogs outside. The selective pressure is melanin protecting against ionising radiation.
Describe the mechanisms of evolution.
Evolution is change in the inherited traits of a population through successive generations: the genetic content of a population changes over time. Mutations are the raw materials of evolution via natural selection.
Do all mutations have selective value?
Not all genetic changes alter phenotype. Some mutations have no effect at all. Some mutations in introns have no effect. Most mutations between genes have no effect.
Describe the ‘Long Term Evolution Experiment’ methods.
Richard Lenski did this experiment in 1988.
Day 1: 2 flasks of E. Coli.
Day 2: 12 flasks innoculated (1/100 dilution) and grown in medium with glucose and citrate.
Day 3: Each flask sub-cultured.
Day 4 +: Every day they were subcultured. Every 75 days, the mean ‘fitness’ is estimated and samples are frozen: a ‘fossil record’: Over 35 years, experiment still running.
Describe the ‘Long Term Evolution Experiment’ timeline and key findings.
In all flasks: growth rate increased and cell size increased.
In some flasks, defects in DNA repair evolved, giving ‘mutator’ phenotypes with elevated mutation rates: these can therefore evolve more rapidly. In one flask, Ara-3, the ability to use citrate in aerobic conditions evolved. Ara-3, evolved a mutator phenotype.
How is citrate used in anaerobic conditions?
Anaerobic conditions: expresses a citrate transporter and can take up citrate from the growth medium.
How is citrate used in aerobic conditions?
Aerobic conditions: citrate transporters are NOT expressed so E. Coli cannot utilise external citrate as an energy source.
Describe the Cit+ strain.
The duplication of the citrate transporter gene. This second copy had de-regulated expression, s citrate transporters were expressed in aerobic growth. Cit+ strains can now utilise citrate as well as glucose as C and energy sources: this gives them a selective advantage.
Define gene flow.
Gene flow: the movement of alleles between previously separate populations. The movement of alleles can be by:
- migration of adults and subsequent mating.
- movements of gametes and subsequent fertilisation.
What are the three main mechanisms if gene flow?
- Genetic drift removes genetic variation within demes (sub-populations) but leads to differentiation between demes, all by random changes in allele frequencies.
- Gene flow introduces new alleles into demes within a metapopulation and by itself can lead to genetic homogeneity between the demes.
- Selection and reproductive isolation.
Define species.
At a eukaryotic level: it’s a population of organisms that can potentially or actually interbreed, giving viable fertile offspring
What is a post genomic era and describe what this brought on.
1995+ is the post-genomic era. The baoratory of J. Craig Venter produced the first complete genome sequence of any organism, the Influenza virus.
What is the human genome project?
Rough drafts were published in 2001, from the publicly funded Human Genome project in nature. The first draft complete sequence was reported on April 14, 2003.
What are the goals of the Human Genome Project?
- Identify all of the genes in human DNA.
- Determine the sequences of the DNA base pairs that make up the human genome.
- Store this information in databases.
- Improve tools for data analysis.
- Transfer related technologies to the private sector.
- Address the ethical, legal and social issues that may arise from the project.
How many (protein-coding) genes do we have? Describe the history behind this.
The first surprise is 100,000 human genes were expected with only 22,000 being protein coding. Only 1.5% of our genome is protein coding.
What are the benefits of the HGP?
It helps understand our evolutionary history, personalised medicine can be predictive and preventative.
Describe ‘The Central Dogma of Molecular Biology’.
Proposed by Francis Crick (1958). It is the transfer of information from DNA via an RNA intermediate to protein. It recognised ‘DNA makes RNA makes protein’. The central dogma also recognises that DNA replication and RNA replication also transfer information.
Who was Gregor Mendel?
The founder of genetics, (1822-1884). He was a friar who demonstrated that the inheritance of certain traits in pea plants follow particular patterns, now referred to as the laws of Mendelian inheritance.
Why did Mendel work with peas?
They produce large numbers of offspring. They have a short generation time. They are able to self-fertilise and cross-fertilise are possible. Pure-breeding lines with contrasting features were available. Only simple tools are needed.
What is ‘The Chromosome Theory Of Inheritance’?
Made in 1902 by Sutton. A genetic theory that states that chromosomes are the physical basis for genetic inheritance. He proposed that Mendel’s factors were carried on chromosomes.
What did Morgan do?
He crossed red eye and white eye flies to research more into inheritance. The eye colour gene is on the X chromosome, but not on the Y.
Describe what ‘one gene-one enzyme’ is.
Beadle and Tatum 1941. They used the red bread mould as a model organism. It can grow rapidly on a very simple medium containing only salts, carbon and nitrogen sources, and biotin (vitamin H). They generated mutants with different missing or altered steps in a biochemical pathway.
What are auxotrophs and prototrophs?
Auxotroph: A mutant that requires a particular additional nutrient.
Prototroph: the normal strain which doesn’t require that nutritional supplement.
Who discovered nucleic acid?
The Swiss physician Friedrich Miescher investigated the nuclei of leukocytes found in pus scraped from bandages. During his experiments, he noticed a substance he called ‘nuclein’ with unexpected properties. It contained carbon, nitrogen and hydrogen like known proteins, but was rich in phosphorus with no detectable suphur.
Describe ‘the discovery of transformation’.
Frederick Griffith 1928 was a british bacteriologist who worked on the epidemiology and pathology of bacterial pneumonia. In 1928 he demonstrated bacterial transformation, where a bacterium changes its form and function through the action of a transforming principle or transforming factor.
Describe X - ray crystallography, a helix and the cross.
The crystalline target molecules diffract X-rays and cause exposed patches on photographic films. The resulting diffraction pattern is a unique ‘signature’ of the molecule.
What did Phoebus Levene?
In 1930, he showed that each building block of DNA is a nucleotide: a phosphate group linked to a deoxyribose sugar - which, in turn, is linked to one of four nitrogenous bases.
Describe the polarity of DNA strands.
DNA strands have polarity: a 5’ end and a 3’ end.
Describe ‘the tetranucleotide model’.
Phoebus Levene came up with the model in 1930. She proposed that the 4 nucleotides occurred in tetranucleotide blocks, with the bases pointing outwards. DNA was therefore simple and repetitive and couldn’t be the genetic material.
Describe the X-ray images of DNA.
DNA couldn’t be crystallised at the time but it could be stretched into long fibres that could be mounted in front of the X ray source: X-ray fibre diffraction. Maurice Wilkins stretched DNA and air dried it. Raymond Goslin, Rosalind Franklin’s PhD student, stretched DNA and left it hydrated.
Describe Watson and Crick’s model.
They came up with this model in 1953. They made it with metal scraps at almost 2m tall. Watson: specific A/T and G/C base pairing scheme. Crick: idea of antiparallel strands. Everything clicking into place beautifully.
What are the 6 key features of the Watson-Crick model?
- Right-handed helix.
- The strands are anti-parallel.
- Bases in: Sugar-phosphate out.
- Complementary Base-pairing.
- Base pair distance.
- Major and Minor grooves.
Generally describe DNA replication.
DNA must be decompacted and then copied faithfully during cell division. The error rate is very low.
What are the 2 predictions that were made from the Watson-Crick model?
- DNA strands are held together by ‘Watson-Crick’ base pairing, consistent with Chargaff’s rules. To make this work, the strands must be anti-parallel.
- Each strand is therefore complementary to the other, so each can act as a template for DNA replication. DNA replication is therefore be semi-conservative.
Describe semi-conservative, conservative and dispersive DNA replication models.
Semi-conservative: Each daughter DNA molecule contains one parental strand and one newly-replicated strand. WATSON-CRICK model.
Conservative: The parent helix is conserved, whilst the daughter helix is completely new.
Dispersive: parent helix is broken into fragments, dispersed, copied and then assembled into two new helices.
How did Meselson and Stahl contribute to DNA replication?
They described caesium chloride equilibrium density gradient centrifugation (technique that separates molecules based on their densities).
How did Arthur Kornberg contribute to DNA replication?
He took E. Coli protein extracts, and worked out the in vitro conditions that were required for this extract to make new DNA:
- A template DNA to copy.
- dATP, dTTP, dCTP, dGTP
- The co-factor Mg2+
- An energy source, ATP
- A small piece of DNA (a PRIMER) with a free 3’ OH.
Describe Pol I and Pol III.
Pol I is NOT the enzyme that is used for replication of E. Coli DNA.
Pol III is essential for DNA replication in E. Coli.
What is the directionality of DNA synthesis?
DNA is synthesized in the 5’ → 3’ direction.
How is DNA replication semi-conservative?
Each new DNA molecule consists of one original strand and one newly synthesized strand.
What are the leading and lagging strands in DNA replication?
Leading strand: Synthesized continuously in the same direction as the replication fork.
Lagging strand: Synthesized discontinuously in fragments, opposite to the replication fork.
What are Okazaki fragments?
Short DNA fragments (~100-200 nucleotides in eukaryotes and ~1000-2000 nucleotides in E. coli) synthesized on the lagging strand.
Why can’t DNA be synthesized in the 3’ → 5’ direction?
5’ → 3’ synthesis allows proofreading and energy-efficient polymerization due to nucleotide hydrolysis.
What is the role of RNA primers in DNA replication?
RNA primers provide the free 3’-OH group needed to initiate DNA synthesis.
Which enzyme synthesizes RNA primers?
DNA primase, a DNA-directed RNA polymerase.
How are Okazaki fragments joined?
DNA ligase uses ATP to seal the nicks between fragments.
What is the “trombone model” in lagging strand synthesis?
A model describing how the DNA polymerase loops the lagging strand to synthesize Okazaki fragments in coordination with helicase.
What is the role of single-stranded DNA-binding proteins (SSBs)?
SSBs prevent ssDNA from reannealing and protect it from nuclease activity during replication.
What causes DNA supercoiling during replication?
The unwinding of DNA at the replication fork causes supercoiling upstream.
How is DNA supercoiling resolved?
Type I topoisomerase: Relaxes negatively supercoiled DNA.
Type II topoisomerase (e.g., gyrase): Converts positively supercoiled DNA into negatively supercoiled DNA.
What are telomeres, and why are they important?
Telomeres are repetitive DNA sequences at chromosome ends, protecting against loss of genetic material during replication.
How does telomerase function?
Telomerase uses an RNA template to extend the 3’ end of the parental lagging strand template.
In which cells is telomerase active?
Germline cells, some epithelial cells, haematopoietic cells, and most cancer cell lines.
What is the correlation between telomeres and cellular ageing?
Telomere shortening limits cell division, contributing to ageing. Telomerase can counteract this process, as seen in cancer cells with “immortal” phenotypes.
How is bacterial DNA replication bi-directional?
Two replication forks move in opposite directions from an origin of replication.
What enzyme separates catenated daughter chromosomes in bacteria?
Topoisomerase IV, a type II topoisomerase.
What is the purpose of DNA sequencing?
To determine the order of nucleotides in a DNA strand.
What was Frederick Sanger’s contribution to sequencing?
He developed the dideoxy (Sanger) sequencing method using dideoxynucleotides as chain terminators.
How does a dideoxynucleotide (ddNTP) work in sequencing?
It terminates DNA strand extension because it lacks the 3’-OH group needed for further elongation.
What are the key steps in Sanger sequencing?
Add template DNA, primer, DNA polymerase, dNTPs, and a ddNTP.
Perform DNA synthesis.
Separate fragments by size via electrophoresis.
Use autoradiography or laser detection to determine the sequence.
How does automated sequencing differ from manual Sanger sequencing?
Automated sequencing uses fluorescently labeled ddNTPs in one reaction tube and detects fragment sizes with lasers.
Who invented PCR, and what is its purpose?
Kary Mullis invented PCR to amplify specific DNA sequences.
What are the essential components of a PCR reaction?
Template DNA, two primers, dNTPs, buffer with MgCl₂, and Taq polymerase.
Why is Taq polymerase critical for PCR?
It is heat-stable, allowing repeated cycles of denaturation, annealing, and extension.
What are the main steps of a PCR cycle?
Denaturation (94°C): Separate DNA strands.
Annealing (45–65°C): Primers bind to target sequences.
Extension (72°C): Taq polymerase synthesizes DNA.
How many copies of DNA are generated after 30 PCR cycles?
Over 1 billion copies of the target DNA sequence.
How is PCR used in prenatal genetic screening?
It amplifies DNA from small samples, like chorionic villus tissue, to detect genetic conditions.
What is an example of PCR in ancient DNA research?
Amplifying Neanderthal DNA from fossils to study human evolution.
How is PCR applied in forensic science?
It amplifies DNA from small samples, such as blood or hair, for DNA profiling.
What are STRs, and why are they important in forensics?
Short tandem repeats (STRs) are DNA regions with variable repeat numbers, used to uniquely identify individuals.
What is multiplex PCR?
A method that amplifies multiple DNA targets in one reaction using specific primers for each region.
How does PCR introduce specific mutations?
By using primers with engineered mismatches near their 5’ ends.
What did the Neanderthal DNA studies reveal about human evolution?
Modern Europeans and Asians have ~2% Neanderthal DNA, suggesting interbreeding.
How does PCR aid in studying archaic human genomes?
It amplifies DNA from fossils, revealing genetic links between ancient and modern humans.
What are some limitations of single STR analysis in DNA profiling?
A single STR has limited discriminatory power, as unrelated individuals may share similar repeats.
How does the CODIS system enhance forensic DNA profiling?
By analyzing multiple STR loci, increasing accuracy and discrimination among individuals.
What are the main stages of the central dogma of molecular biology?
DNA replication: DNA is copied for cell division.
Transcription: DNA information is transcribed into RNA.
Translation: RNA is translated into protein.
Reverse transcription and RNA replication are additional processes in some contexts (e.g., retroviruses).
What is transcription, and how does it differ from DNA replication?
Transcription is the process of synthesizing RNA from a DNA template. It differs from replication in that only one strand of DNA (the template strand) is copied, and uracil (U) replaces thymine (T) in RNA.
What is the difference between the sense and antisense strands of DNA?
Sense strand: The non-template strand; its sequence matches the RNA transcript (except for T/U substitution).
Antisense strand: The template strand; RNA polymerase reads this strand to create a complementary RNA molecule.
What is the composition of bacterial RNA polymerase?
Core enzyme: Composed of subunits α (2 copies), β, β’, and ω.
Holoenzyme: Core enzyme plus σ subunit, which directs the polymerase to specific promoters.
What is a bacterial promoter, and what are its key features?
A promoter is a DNA sequence that initiates transcription.
Key regions:
-35 sequence (TTGACA): Recognized by RNA polymerase.
-10 sequence (TATAAT): Facilitates DNA unwinding.
Transcription starts at the +1 site, often an A or G.
How do σ factors affect RNA polymerase function?
Different σ factors guide RNA polymerase to specific sets of promoters, allowing bacteria to regulate transcription based on environmental conditions.
What are the three stages of bacterial transcription?
Initiation: RNA polymerase holoenzyme binds to the promoter, opens the DNA helix, and starts RNA synthesis.
Elongation: RNA polymerase synthesizes RNA, forming a transcription bubble.
Termination: Transcription ends, and RNA polymerase dissociates from DNA.
What is the transcription bubble, and how does it form?
The transcription bubble is an unwound region of DNA (~17 bp) where RNA polymerase synthesizes RNA. It forms when RNA polymerase pulls DNA downstream and unwinds it without requiring ATP.
How does RNA polymerase ensure accuracy during transcription?
RNA polymerase can backtrack and remove misincorporated ribonucleotides. However, its error rate is higher than DNA polymerase because RNA errors are not passed to progeny.
What are the two mechanisms of transcription termination in bacteria?
Rho-independent termination: Involves a GC-rich hairpin structure in RNA followed by a U-rich sequence. The hairpin disrupts RNA-DNA pairing, releasing RNA.
Rho-dependent termination: Requires the rho protein, a helicase that disrupts the RNA-DNA hybrid when it catches up to RNA polymerase.
How does the rho protein function in transcription termination?
Rho binds to a C-rich, G-poor region of the RNA and uses ATP hydrolysis to move along the RNA, eventually dissociating the RNA from the DNA template.
How is transcription regulated in bacteria?
Transcription is regulated by:
- Promoter strength (closeness to consensus sequences).
- Specific σ factors that recognize distinct promoter sets.
- Presence of operator sequences that control RNA polymerase access.
What is rifampicin, and how does it affect bacterial transcription?
Rifampicin is an antibiotic that binds to RNA polymerase’s RNA exit channel, preventing initiation but not affecting elongation by already active RNA polymerase.
Why does RNA polymerase bend DNA during transcription?
Bending the DNA helps destabilize the double helix, making it easier to unwind and initiate transcription.
Why is uracil not found in DNA?
Cytosine can spontaneously deaminate to form uracil. If uracil were in DNA, it could cause mutations by pairing with adenine, so DNA repair mechanisms replace uracil with cytosine.
How do bacterial and eukaryotic transcription differ in cellular compartmentalization?
Bacteria: Transcription and translation occur in the same compartment and may be coupled.
Eukaryotes: Transcription occurs in the nucleus, and translation occurs in the cytosol, so they are uncoupled.
What are the key differences between bacterial and eukaryotic mRNA?
Bacterial mRNA: Often polycistronic (encodes multiple proteins) and used directly for translation.
Eukaryotic mRNA: Monocistronic (encodes one protein) and undergoes extensive processing before translation.
How many RNA polymerases are there in eukaryotic cells, and what are their functions?
RNA Pol I: Synthesizes rRNA.
RNA Pol II: Synthesizes mRNA and some snRNAs.
RNA Pol III: Synthesizes tRNA and some small RNAs.
What is unique about RNA polymerase II?
RNA Pol II has a C-terminal domain (CTD) that is phosphorylated to recruit processing enzymes for capping, splicing, and polyadenylation of mRNA.
What are the conserved elements in RNA Pol II promoters?
TATA box: A TATA-rich sequence, located ~ -30 to -100 bp upstream of the start site.
Inr (initiator): Located at the transcription start site.
DPE (downstream promoter element): Found +28 to +32 bp downstream in TATA-less promoters.
CAAT box/GC box: Found between -40 and -150 bp upstream.
How does TFIID function in transcription initiation?
TFIID binds to the TATA box via its TBP subunit. It acts as a nucleus for assembling the basal transcription apparatus, including TFIIA, TFIIB, RNA Pol II, TFIIF, TFIIE, and TFIIH.
What roles does TFIIH play in transcription?
Helicase activity: Opens the DNA helix.
Kinase activity: Phosphorylates RNA Pol II’s CTD, transitioning it from initiation to elongation.
How is DNA bending important in transcription?
Binding of TBP to the TATA box bends the DNA, widening the minor groove, which helps recruit other transcription factors and opens the DNA for transcription.
What is the role of enhancers in transcription?
Enhancers increase transcription levels, even from a distance (several kb away). They interact with the transcription initiation complex via DNA looping mediated by proteins and cofactors.
What is combinatorial control of transcription?
Transcription is regulated by the presence of specific activators that bind enhancers. This ensures cell-type specificity, such as immunoglobulin expression in B cells or crystallin expression in eye lens cells.
How is the 5’ cap added to eukaryotic mRNA?
A triphosphate group at the 5’ end of the RNA is modified to a 5’-5’ triphosphate linkage with GTP.
The terminal guanine is methylated, forming a cap structure that stabilizes mRNA and enhances translation.
What is the function of the poly(A) tail?
The poly(A) tail, added at the 3’ end, stabilizes mRNA and regulates its degradation. When the tail becomes short enough, the mRNA is degraded.
What is splicing, and why is it important?
Splicing removes introns and joins exons in pre-mRNA.
It allows the formation of mature mRNA and permits alternative splicing, generating protein diversity.
How does RNA Pol II terminate transcription?
The RNA transcript is cleaved at a poly(A) site by cleavage factors.
The RNA fragment attached to Pol II is degraded, causing Pol II to disengage from DNA.
What is α-amanitin, and how does it affect transcription?
α-Amanitin: A toxin from Amanita phalloides that inhibits RNA Pol II.
It binds the active site, reducing RNA production to a few nucleotides per hour, leading to liver and kidney failure if ingested.
How was the existence of introns discovered?
Introns were discovered using R-loop analysis. By hybridizing mRNA to DNA, researchers found multiple loops of single-stranded DNA that did not align with the mRNA, indicating non-contiguous DNA sequences (introns).
What are introns and exons?
Introns: Non-coding sequences removed during RNA splicing.
Exons: Coding sequences that remain in the mature RNA.
Are introns common in all organisms?
Introns are almost universal in vertebrates but rare in yeast. In humans, more DNA is devoted to introns than exons.
What are the four classes of introns?
Group I introns: Self-splicing, found in rRNA genes of some unicellular eukaryotes and organelles.
Group II introns: Self-splicing, found in fungal and plant organelles.
Spliceosome-dependent introns: Found in nuclear mRNA.
tRNA introns: Spliced by a unique enzymatic mechanism.
What is the mechanism of Group I intron self-splicing?
The intron folds and uses a guanosine (GMP, GDP, or GTP) as a cofactor.
The 3’-OH of guanosine attacks the 5’ splice site, releasing the intron.
A second transesterification reaction fuses the exons.
How does Group II intron splicing differ from Group I?
Group II introns fold into a tertiary structure that brings splice sites together. They use a branch site for lariat formation and involve two transesterification reactions like spliceosome-mediated splicing.
What is the spliceosome, and what is it composed of?
The spliceosome is a large complex responsible for nuclear mRNA splicing.
It consists of small nuclear ribonucleoproteins (snRNPs) like U1, U2, U4, U5, and U6, each containing snRNA and protein subunits.
How is spliceosome assembly coordinated?
U1 binds the 5’ splice site.
U2 binds the branch site.
A U4/U6/U5 trimer joins to form an inactive spliceosome.
U6 and U2 base-pair to activate splicing.
How does the spliceosome facilitate efficient splicing?
The spliceosome provides a structured framework. U6 snRNA base-pairs with U2 and the branch site adenine, creating a bulge for efficient transesterification reactions.
Why is RNA splicing important for eukaryotes?
Splicing ensures proper removal of introns, allows alternative splicing for protein diversity, and coordinates with transcription for efficient gene expression.
What is alternative splicing, and why is it significant?
Alternative splicing allows a single gene to produce multiple protein isoforms by selectively including or excluding exons.
It is developmentally regulated and increases protein diversity, as seen in the calcitonin gene producing calcitonin and CGRP.
What are the “intron-early” and “intron-late” hypotheses?
Intron-early hypothesis: Introns are ancient and played a key role in early evolution, possibly aiding exon shuffling.
Intron-late hypothesis: Introns are later additions, with some encoding homing endonucleases for spreading.
What is exon shuffling, and how do introns enable it?
Introns provide space for recombination without disrupting coding sequences, allowing exons to be rearranged and creating new proteins with novel functions.
How do mutations in splicing contribute to genetic diseases?
Mutations can destroy splice sites or create aberrant ones, leading to incorrect splicing. About 15% of genetic diseases, such as certain cancers and inherited disorders, are caused by splicing errors.
What is an example of a gene that demonstrates the complexity of splicing?
The human dystrophin gene, which is 99.5% introns and takes 13-17 hours to transcribe, highlights the metabolic cost and functional importance of splicing.
Describe what coding happened in 1950s.
A string of nucleotides, in a 4-letter alphabet, encoded in a double helix was somehow transcribed into a working copy which was somehow translated into a string of amino acids, with a 20-letter alphabet.
What did George Gamow do?
In 1954, he formed the ‘RNA Tie Club’. 20 hand picked members, one for each amino acid. He deduced that the minimal coding unit must be a triplet, the code must be degenerate and this led to the ‘Diamond hypothesis’.
Describe what the diamond code is.
Each amino acid would fit directly into distinct diamond shaped pockets formed within the grooves of DNA where the 4 sides of each pocket would be defined by the 4 bases.
