Genes/DNA/RNA/Protein Flashcards
Describe the initiation stage in transcription
- Activator proteins bind to a DNA sequence called enhancers. Then DNA bends and it comes into contact with promotor region
- Transcription factors and RNA polymerase join activator proteins binds to promoter region, creating the transcription initiator complex
Describe the elongation stage in transcription
- RNA polymerase unzip DNA helix and starts to build mRNA from the 5’ to 3’ direction
- Promoter region does not get transcribed
- Polymerase puts together the complimentary ribonucleotides to build complementary mRNA strand according to template strand
Which strand is the template strand and which strand is the coding strand
Template strand is the 5’ to 3’ strand, which is used as a guide to build the mRNA. The coding strand is the 3’ to 5’ strand, it is identical to the mRNA except it contains uracil instead of thymine.
Describe the termination stage in transcription.
- Once RNA polymerase reaches the termination sequence, it signals the end of the gene being transcribed. Or sometimes termination ends randomly.
- mRNA dissociates from gene and the mRNA is the primary transcript.
What is capping and tailing?
They are post-transcriptional modification to the primary transcript before it leaves the nucleus into the cytoplasm
1. Capping is the addition of a 5’ cap that contains 7 methyl guanosine. It protects mRNA from digestion by phosphates and nucleases.
2. Tailing is the addition of 200-300 adenine ribonucleotides to the 3’ end by poly-A-polymerase to create a poly-A-tail.
What is splicing?
It is a post transcriptional modification. Gene have exons and introns that are interspersed between each other. If introns are translated, protein will not fold properly. Spliceosomes cut introns out and join extrons together.
Why is spinal muscular atrophy (SMA) caused by splicing problems?
People with SMA has lower levels of survival of motor neuron (SMN) proteins. SMN1 and SMN2 genes are nearly identical and SMN1 usually used to make SMN proteins. However, people with SMA have nonfunctional SMN1 and rely on SMN2. SMN2 has mutation that blocks a protein from properly splicing exon 7 into the mRNA. Shorter mRNA produces shorter SMN protein that does not function.
Describe the process of translation
- mRNA enters cytoplasm and ribosome and two subunits binds to the 5’ end and acts as the construction site for polypeptides.
- Ribosome move from 5’ to 3’ direction and tRNA adds an amino acid (anticodon) every time ribosome reads a codon.
- Two sites for tRNA. Acceptor site and Peptide site. Bring first amino acid to P site first (start codon) and then bring second amino acid to A site.
- The amino acid from P site bonds to the amino acid in A site , and then ribosome shifts over one codon and the second amino acid is now in P site. Third amino acid enters A site. Polypeptide chain continues to grow.
- Repeat until ribosome reaches stop codon where no tRNA exists for it.
- Release factor dismantles complex and release the chain from ribosome.
Explain cDNA microarray
- Take mRNA from different stages/or samples
- Reverse transcriptase into cDNA and then flourescently label the nucleotides
- Hybridisation: have the DNA recombine with its complementary sequence on the micro array and see which colour expressed more of which gene to determine gene expression profiles
What does CRISPR stand for?
Clustered, regularly, interspaced, short, palindromic, repeats
How does CRIPSR work?
- Produced by bacteriophage when a virus attacks
- Form Cas 9 complex: guide RNA matches the virus DNA sequence, tracer RNA that binds with cas 9, and cas 9 protein.
- Cas 9 binds to 2-6 base sequence of virus DNA (PAM), unzips dna, guide RNA matches the sequence, and cas 9 cuts out the sequence, disabling the virus
Applications of CRISPR
- Gene editing
- Used in living cell to cut out desired DNA sequence by changing guide RNA to match target. Even as cell try to repair DNA, it is error prone and will disable the cell.
- Can replace mutated sequence with desired DNA in stem cells and fertilised egg - Promote gene transcription
- Gene silencing
- Attaching fluorescent proteins to visualise genome or follow chromosome position in nucleus
Clinical Trials in Gene Therapy
- Eye therapies (ex. in vivo; leber congenital amaurosis, responsible for cilia function in retina, results in vision loss caused by point mutation at CPEP290)
- Sickle cell and beta thalassaemia
- Ex vivo cancer immunotherapy
- SMA gene therapy
- Chimeric antigen receptor (CAR) T-cell therapy: using immune T cells to fight cancer by changing them to find and destroy cancer cells
What is a challenge of CRISPR
Chromothripsis: chromosome shattering
Describe DNA (deoxyribonucleic acid)
- B DNA structure most common
- Major and minor grooves
- Phosphate, pentose sugar, nitrogenous base
Difference between DNA and RNA
DNA
- H at carbon 2
- Thymine
- Double stranded
RNA
- OH at carbon 2
- Uracil
- Single stranded
What does degenerate mean
Several codons code for the same amino acid
What are histones and how do histones affect gene expression
Histones are a group fo 8 proteins of two of each H2A, H2B, H3, H4. Each has 200 nucleotides wrapped around it (nucleosome). Post translational modifications to histone structure control which part of DNA is exposed and affects which genes get transcribed.
What are transcription factors and how do they affect gene expression
- Activator proteins bind to enhancer region that causes DNA to bend and contacts promoter region and transcription factors to make it easier for RNA polymerase to attach
Activator protein-> contact promoter region -> RNA polymerase binds
- Insulators are regions that can stop enhancers from binding to the promoter if CTCF protein binds to it
- Methylation to C nucleotides prevents CTCF from binding
Why is genetic variation important?
- Genetic variation describes DNA diff between people
- Explains for phenotypes
- Includes diseases/health issues
- Interaction with environment contributes to phenotype
Copy number variation
Gains and losses of whole/arms of chromosomes
Strutural variations
- From 1 kilobase to 3 megabases in size
1. Deletion: Portion of chromosome deleted - Ex. Facioscapulohumeral Muscular Dystrophy (FSHD) is a deletion of D4Z4, which activates DUX4 gene, causing muscle toxicity
2. Duplication: Part of chromosome duplicated
3. Translocation: Part of one chromosome broken off and attached to another chromosome
4. Inversion: Portion of genetic material flipped in opposite order
Sequence level variation
- SNP Single Nucleotide Polymorphism: single base pair change
- Insertion/deletions (indels): if it affects more than one base
How many bases does everyone have that differ from reference genome
4 to 5 million
