WEEK 3 (CRISPR and non-coding RNAs) Flashcards
The CRISPR-Cas9 system in bacteria is akin to our body’s _________________
Immune system
CRISPR refers to repeated located in the ___________
Bacterial DNA
The CRISPR sequences are recognised by _______________
Guide RNA
What is the name of the chemical reaction that catalyses the cleavage of a peptide bond?
Hydrolysis
A codon contains how many nucleotides?
3
Which position of a codon is said to wobble?:
Third
The genetic code translated the language of _____________
RNA into that of proteins
The genetic code is
Universal except for rare exceptions in mitochondria and some protozoa
Which of the following has been used an evidence that primitive life forms lacked both DNA and enzymes?
RNA can both code genetic information and act as a catalyst
Codon that specify the amino acids often differs in the ____________
Third base
The codon which do not specify an amino acid are called?
Termination codons
In prokaryotes, AUG encodes __________
N-formyl methionine
Translation begins ______________
At the start codon
To which of the following does thymine form hydrogen bonds in DNA?
Adenine
What role does messenger RNA play in the synthesis of proteins?
It provides the genetic blueprint for the protein
What role does small nuclear RNA play in the synthesis of proteins?
It modifies messenger RNA molecules prior to protein synthesis
What type of enzyme is used in recombinant DNA technology to split a specific sugar phosphate bond in each strand of a DNA double helix?
Restriction enzyme
An esterase enzyme cleaves ester bonds. The restriction enzyme cleaves sugar phosphate bonds in DNA. The lipase enzyme breaks down fats. The ligase enzyme reforms sugar phosphate bonds after annealing.
The word homologous literally means same location. How does this relate to homologous chromosomes?
The bands resulting from staining are found in the same location and the chromosomes have the same genes in the same location
What is the characteristic of the coding capacity of codons?
61 sense codons determine the 20 amino acids & 3 of the codons do not code for any amino acid
In protein synthesis in prokaryotes ____________
The initiating amino acid is N-formyl methionine
How do the daughter cells at the end of mitosis and cytokinesis compare with their parent cell when it was in G1 of the cell cycle?
The daughter cells will have half the number of chromosomes and half the amount of DNA
What are stRNAs?
Small temporal RNA and a form of microRNA
What are scnRNAs?
Produced and function by an RNAi-related mechanism
What are microRNAs?
A family of molecules that helps cells control the kinds and amounts of proteins they make thus helping to control gene expression
Where are molecules of microRNA found?
In cells and in the bloodstream
Describe the processing of microRNA
1) microRNA gene is activated
2) DNA strand opens up
3) Gene is copied/transcribed in the form of RNA
Describe what happens after microRNA is processed in the nucleus
Initial gene transcript is called primary miRNA (pri-miRNA) -> In the cell nucleus, these hairpin-loop molecules are cut to form DOUBLE STRANDED precursor miRNA (pre-miRNA) -> pre-miRNA is transported to the cytoplasm
What is the link between microRNA and cancer?
MicroRNA can be damaged by mutations and a mutation in microRNA gene can leave the cell without that particular microRNA or reduce it to a low level in the cell -> Abnormally low levels of a microRNA can lead to over expression of genes that microRNA regulates -> Cancer development and progression
Describe the formation of miRNAs
1) Derived from SINGLESTRANDED PRECURSOR RNAs that contain COMPLEMENTARY SEQUENCES that allow them to fold back on themselves to form a double-stranded RNA with a stem-loop at oe end
2) This pseudo-dsRNA/pri-miRNA is cleaved at a specific site near its TERMINAL LOOP by DROSHA (an endonuclease) to generate a pre-miRNA that has a 3’ overhang at one end
Describe the action of miRNAs
1) The pre-miRNA is exported to the cytoplasm where it is cleaved by DICER into a small duplex miRNA that has a 3’ overhang at both ends
2) The double-stranded RNA becomes associated with a protein complex containing an ARGONAUTE PROTEIN (usually Ago1) leading to the separation of the strands and removal of the passenger strand (miRNA)
3) The single-stranded guide miRNA binds to a complementary region on an mRNA and inhibits translation of the message (or DEADENYLATION and DEGRADATION of the mRNA)
What differentiates siRNA and miRNA?
- Unlike siRNAs, miRNAs that inhibit translation are only partially complementary to the target mRNA
- siRNAs are produced by any DOUBLE STRANDED RNA in the cell (viral genome)
- piRNAs are encoded in the HOST GENOME and provide the host with a genetically encoded defence ready to attack any transcript with a similar sequence
Germ cells of animals express a distinct class of which small RNAs?
piwi-interacting RNAs (or piRNAs) that suppress the movement of transposable elements in the germ line
How does the movement of transposable elements pose a threat to the genome?
It can disrupt the activity of a gene into which the element happens to insert
When would movement of transposable elements have minimal and maximal effect?
MINIMAL: If GENOMIC JUMPING were to occur during the life of an adult liver or kidney cell the consequences would be MINIMAL because the transposition event affects only that particular cell and its daughters
MAXIMAL: If TRANSPOSITION EVENT happened in a GERM CELL, a cell that has the capability of producing gametes, then the event has the potential to affect every cell in an individual of the next generation
What are the properties of piRNAs?
- Associate with proteins called PIWIs
- Subclass of the ARGONAUTE FAMILY
Where have the roles of PIWI proteins been studied?
In fruit flies where deletion of these proteins leads to defects in the suppression of TRANSPOSON MOVEMENT in germ cells and ultimately to failure of gamete formation
What are the important differences between piRNAs and si/miRNAs?
- piRNAs are longer than the other small RNAs (24-32 nucleotides)
- Mammalian piRNAs can be mapped to a small number of huge GENOMIC LOCI
- piRNAs can be subject to an AMPLIFICATION process that generates additional copies of piRNAs
- Formation of piRNAs does not involve the formation of dsRNA precursors or cleavage by DICER RIBONUCLEASE, instead it depends on ZUCCHINI (a single-stranded RNA endonuclease)
Describe the function of Zucchini
Acts as LONG, SINGLE-STRANDED PRIMARY TRANSCRIPT to release the short piRNAs. Those piRNAs that are active in the cell against transposable elements are then amplified in a subsequent step.
Where are piRNA precursors produced?
From genetic regions named piRNA clusters which can be divided into UNI-STRAND or DUAL-STRAND CLUSTERS & some from the 3’ UTR or protein-coding genes or from individual TRANSPOSONS
What is the difference between Uni-strand clusters and Dual-strand clusters?
- UNI-STRAND CLUSTERS = give rise to precursors mapping only to one strand
- DUAL-STRAND CLUSTERS = produce precursors mapping to both genomic strands
What is the difference between Uni-strand and Dual-strand cluster transcription?
- UNI-STRAND CLUSTERS have DISTINCT PROMOTER REGIONS and generate 5’-methyl-guanosine-capped and 3’-terminated precursors from one direction
- DUAL-STRAND CLUSTERS have NO DISTINCT PROMOTER REGION and generate NON-POLYADENYLATED piRNA precursors from two directions
What are Dual-strand clusters transcribed into?
piRNA precursors
What happens to piRNA precursors once transcription is finished?
piRNA precursors are transported out of the nucleus
What is similar between siRNA and piRNA?
Both siRNA and piRNA evolved as defence against dangerous genomic elements (viral genomes or transposons)
What are CRISPRs?
Genomic loci containing short DNA segments that are identical to sequences from the DNA of bacteriophage viruses separated by a repeated DNA sequences
CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats
Used as part of an antiviral system
How does CRISPR work?
1) The Cas9 protein forms a complex with GUIDE RNA in a cell
2) This complex attaches to a matching genomic DNA sequence adjacent to a spacer
3) The Cas9-RNA complex cuts the double strands of DNA
4) Programmed DNA may be inserted at the cut
What are Cas (CRISPR-associated) genes?
Accompanying CRISPR are genes that are always located nearby. Once activated, these genes make enzymes that have the ability to act as “molecular scissors” that can cut into DNA.
What are the three characteristics that help CRISPR-Cas9 tech do its function?
- GUIDE RNA (a piece of RNA that locates the targeted gene)
- CRISPR-associated protein 9 (Cas9) (molecular scissors that snip undesired DNA out)
- DNA (desired piece of DNA that is inserted after the break)
Describe how Sickle Cell Anaemia can be treated using CRISPR
1) Patient has sickle cell disease caused by a single DNA letter mutation
2) In lab, scientists create a strand of GUIDE RNA that matches the mutated DNA sequence and Cas9 is added to the RNA mix
3) Patient receives injection of RNA & Cas9 mix
4) Guide RNA identifies the mutated DNA that causes sickle cell disease & Cas9 cuts out the affected sequence
5) Scientists insert a healthy DNA sequence to replace the clipped sequence
What are the properties of Heterogenous nuclear RNA (hnRNA)?
- Immediate product of gene transcription
- Nuclear product is variable in size and is very large
- 75% degraded in the nucleus & 25% is processed to mature mRNA
What did George Gamow propose?
- Each amino acid in a polypeptide is encoded by three sequential nucleotides
- The genetic code is overlapping
How can you distinguish if the genetic code is overlapping and non-overlapping?
If the code is OVERLAPPING then the ribosome would move along the mRNA one nucleotide at a time, recognising a new codon with each move
If the code is NON-OVERLAPPING each nucleotide along the mRNA would be part of only one codon
How could you find out if the genetic code was overlapping or non-overlapping?
From studies of mutant proteins (e.g mutant haemoglobin responsible for sickle cell anemia)
What are the characteristics of the genetic code?
- TRIPLET NATURE (codons & 64 different combinations)
- DEGENERATE (one amino acid is coded by more than one codon)
- NON-OVERLAPPING
- COMMALESS (no signal to indicate the end of one codon and the beginning of the next)
- NON-AMBIGUOUS (a particular codon will always code for the same amino acid)
- UNIVERSAL
- POLARITY (always read in a fixed direction)
- CHAIN INITIATION CODONS
- CHAIN TERMINATION CODONS
Describe the structure of tRNAs
- Between 73 and 93 nucleotides
- Contain significant amount of unusual bases incorporated post transcriptionally
- Sequences of nucleotides in one part of the molecule that were complementary to sequences located in other parts -> tRNAs become folded to form a cloverleaf shape
- Unusual bases concentrated in the LOOPS disrupt HYDROGEN BOND FORMATION in these regions and serve as potential RECOGNITION SITES for various proteins
- All mature tRNAs have triplet sequence CCA at their 3’ end
- Constructed of two double helices arranged in the shape of an L
How is the triplet CCA at the 3’ end in tRNAs added in prokaryotes and eukaryotes
In PROKARYOTES the three nucleotides are encoded in the tRNA gene whereas in EUKARYOTES they are added enzymatically
What is tRNA charging?
During protein synthesis when each transfer RNA molecule is attached to the correct amino acid
Amino acids are covalently attached to the 3’ ends of their cognate tRNA by which enzyme?
aminoacyl-tRNA synthetase
How many different aminoacyl-tRNA synthetases do organisms usually contain?
20
One for each of the 20 amino acids
Describe the two-step reaction that Aminoacyl-tRNA synthetases carry out
1) Energy of ATP activates the amino acid by formation of an ADENYLATED AMINO ACID which is bound to the enzyme
2) Enzyme transfers its bound amino acid to the 3’ end of a cognate tRNA
Describe the movement of ribosomes during translation
Each of the ribosomes initially assemble from its subunits at the initiation codon and then moves from that point towards the 3’ end of the mRNA until it reaches a termination codon. As each ribosome moves away from the initiation codon, another ribosome attaches to the mRNA and begins its translation activity.
Describe the Exon-junction complex (EJC)
When an intron is removed by a SPLICESOME, a complex of proteins is deposited on the transcript 20-24 nucleotides UPSTREAM from the newly formed exon-exon junction. This stays with the mRNA until it is translated; if the EJC stays until the ribosome finishes translation it is marked for DEGRADATION by RIBONUCLEASES.
What would happen during translation of an mRNA that contained a premature termination codon?
The ribosome would stop at the site of the mutation and then dissociate, leaving any EJCs that were attached to the mRNA downstream of the site of premature termination. These remaining EJCs set in motion a series of events leading to the enzymatic destruction of the abnormal message.
What is Tay-Sachs disease?
A rare inherited disorder that progressively destroys nerve cells in the brain and spinal cord and is caused by a mutation in the HEX A gene. HEX-A gene provides instructions for making part of an enzyme called B-hexosaminidase A which plays a critical role in the brain and spinal cord.
[B-hexosaminidase A helps break down a fatty substance called GM2 ganglioside]
CYTOGENETIC LOCATION: 15q23 (Long arm chromosome 15)
SYMPTOMS:
- Nerve cells destroyed in brain and spinal cord
[Symptoms appear 3-6 moths after birth]
- Loss of motor control
- Atrophy of muscles
- Seizures
- Death
