6/7: epigenetics/RNA Flashcards
(34 cards)
What is the dogma of DNA transcription?
- DNA is used as a template for the transcription of mRNA; mRNA exits the nucleus and is translated by ribosomes to to synthesize proteins (translation)
- however, genetic information changes going from DNA-mRNA-protein
How was DNA discovered?
- Rosalind Franklin began training Raymond Gosling on X-ray diffraction technique
- DNA existed in two forms:
1) the dry A form, which held less water
2) the wet B form, in which water molecules cling to the DNA, causing it to stretch out
- DNA existed in two forms:
- Watson and Crick’s initial model was wrong because Watson did not take notes while Franklin was telling him about her X-ray crystallography results
- Goslin passed Franklin’s image on to Watson and Crick; Watson and Crick happened to know someone on the Medical Research Council who had a copy of Franklin’s report and was willing to show it to them (these are confidential)
What are the major features of DNA (structure, bonding, directionality, etc)?
- a polymer of four nucleotides, each having three components: a nitrogenous (nitrogen-containing) base, a pentose sugar called deoxyribose, and a phosphate group; nitrogenous base: purines (two organic rings: adenine & guanine) and pyrimidines (one organic ring: thymine & cytosine)
- the amount of A is always equal to the amount of T and C is always equal to G (this ratio varies from species to species)
- the two strands connected by hydrogen bonds between nitrogenous bases; adenine can form two hydrogen bonds with thymine and only thymine (weaker); guanine forms three hydrogen bonds with cytosine and only cytosine (stronger)
- Nucleotides are linked to each other by their phosphate groups, which bind the 3’ OH end of one sugar to the 5’ (phosphate group) end of the next sugar; the DNA double helix is anti-parallel, which means that the 5’ end of one strand is paired with the 3’ end of its complementary strand (and vice versa)
- the outer edges of the nitrogen-containing bases are exposed and available for potential hydrogen bonding; these hydrogen bonds provide easy access to the DNA for other molecules, including proteins, that play vital roles in the replication and transcription of DNA
What is histone deactelyase (HDAC)?
- HDAC remove acetyl groups and when they’re attached to histone proteins, you can’t acetylate
- TF can remove HDAC which can then allow acetyl groups to be added to that histone
What is the pre-initiation complex?
– composed of general transcription factors (GTFs) and RNA Pol II
What are spliceosomes and how do the work?
- Spliceosome is a large complex of proteins that carries out splicing by removal of introns and legation of exons
- Each spliceosome is composed of five subunits called snRNPs (for small nuclear ribonucleoparticles, and pronounced “snurps”.)
- Each snRNP is itself a complex of proteins and a special type of RNA found only in the nucleus called snRNAs (small nuclear RNAs)
- Spliceosome cleaves the sugar phosphate backbone at the GU that starts the intron and then covalently attaches that GU to an internal AG nucleotide within the intron.
- The spliceosome connects the 3′ end of the first exon to the 5′ end of the following exon, cleaving the 3′ end of the intron in the process.
- This results in the splicing together of the two exons and the release of the intron in a lariat form.
What is transcription and transcription factors?
- transcription: the synthesis of RNA using DNA as a template
- transcription factors: regulate changes in gene expression; transcription factors are generally nuclear and can either be constitutively expressed within the cell meaning they are always there but are not active unless phosphorylated (present under basal conditions, for example CREB) or themselves inducible (for example AP-1 (made from Fos and Jun proteins)); 5 – 10% of expressed sequences in human genome encode transcriptional regulators; binds to its specific region more upstream than the promoter region (DNA motif) and affects transcription of specific genes by recruiting co-activators and ATP-dependent nucleosome-remodeling complexes (SWI/SNF)
What is alternative splicing?
– About 95% of human genes undergo alternative splicing.
– Alternative splicing increases the number of mRNA products any one gene can produce, as it allows for skipping exons or mixing and matching exons into mRNA.
– That, in turn, begets a different function of the protein the mRNA codes for
– If a gene contains 6 exons, versions of the mRNA transcribed from that gene might contain
Exons 1-6.
Exons 1,3,5,6
– Produce different forms of a protein from the same gene (ex. five different dopamine receptors that come from the same gene)
– The different forms of the mRNA are called transcript variants, splice variants, or isoforms
What is heterochromatin and euchromatin?
- Heterochromatin: transcriptionally silent genes have dense DNA methylation on DNA and a closed chromatin structure
- Euchromatin: Transcriptionally active genes that have open chromatin structure with histone modifications (acetylation (Ac)) & interaction with euchromatin proteins (ATP-dependent unwinding enzymes) that allow access to the general transcription factors and various coactivator proteins
- these two types of chromatin compaction can be influenced by a wide range of processes including modification to both histones and DNA and ATP-dependent chromatin remodeling complexes
What are activators, co-activators, general transcriptional factors (GTF)?
- activators are a type of transcription factor that increases the transcription of that gene. (ex. CREB)
- co-activators: bind to activators to help increase transcription of a gene by facilitating assembly of pre-initiation complex (ex. HAT)
- general transcription factors (GTF): a class of protein transcription factors that bind to specific sites (promoter) on DNA so RNA poly II can then bind to the gene and transcribe it; pull aside the strands of DNA and move Pol II into elongation mode
What is pre-mRNA, mRNA, and miRNA?
– During transcription, the target gene is copied into a precursor mRNA (pre-mRNA), which includes exons and introns
» introns as intervening sequences that form non-coding RNA (miRNA)
» exons as expressed sequences that form mRNA
– RNA splicing: During the post-transcriptional processing of pre-mRNA (primary transcription), introns (intervening sequences) are removed and exons (expressed sequences) joined and rearranged to form a contiguous coding sequence (mRNA); this changes their sequence and the relative amount of protein
– Noncoding RNA (miRNA): introns that had been removed from pre-mRNA that can go back and alter post/pre transcriptional processing (regulatory functions); can bind to complementary target sites in mRNA which can cause translation repression or degradation of target mRNA genes
What is histone-acetyl transferase (HAT), histone methyl-transferase (HMT)/DNA methyl-transferase (DNMT)?**
- HAT: brings acetyl groups to the histones causing DNA to loosen up around the histones
- HMT: brings methyl groups to the histones causing the DNA to tighten around the histones
- DNMT: brings methyl group to the C5 position of cytosine causing the recruiting of proteins involved in gene repression or by inhibiting the binding of TF
How do antidepressants help with neuronal growth?
- BDNF keeps neurons alive, help spines come out/pull in, wide dendritic fields (BDNF gets bumped up by exercise, learning, etc)
- The gene for BDNF can be modified by chromatin remodeling (epigenetic changes) that affect levels of gene expression
- Stress (psychological or physical) results in methylation of BDNF genes, reducing expression of it BDNF
- Neurons atrophy in the absence of BDNF
- Antidepressants reverse suppression of BDNF expression by downregulate histone deacetylase (HDAC) activity and thus increasing histone acetylation.
How can HDAC be used in cancer treatment?
– Activation of HDAC enzymes by pharmacological intervention is effective in the treatment of some cancers (Why?) –cell replication is stopped
What are chromatin/nucleosome remodeling complex?
- Acetylation disrupts chromatin structure, but it does not expose DNA within nucleosome core particles
- chromatin remodeling complexes (unlike HDAC, HAT, HMT, etc) are not enzymes and are ATP-dependent multi-protein complexes that mediate conformational changes by exposing (or closing) individual DNA sequences
- Ex. SWI/SNF opens up promoter region where general transcriptional factors and RNA Pol II bind to turn on gene transcription (by transcribing DNA to pre-mRNA)
How does DNA methylation inhibit transcription?**
- DNA methylation can inhibit transcription either directly, by blocking the binding of transcriptional machinery to DNA, or indirectly, via methyl-CpG binding domain proteins (MBDs)
- Methyl binding domain proteins (MBD) regulate the condensation of chromatin structure and recruit HDACs and DNMTs, which may further enzymatically modify chromatin components
- MBD activity can reduce transcription of the gene encoding BDNF and attenuate synaptic plasticity
What is a function of chromatin?
– One function of chromatin is to package DNA into chromosomes and control transcription (expression) through opening and closing of its structure (euchromatin/heterochromatin)
What is the difference between genes, chromosomes and chromatin?
- DNA is organized into sequences – genes - that contain information to code for proteins
- DNA and histones are known as nucleosomes, which together form the chromatin structure; the basic unit of chromatin organization is the nucleosome, which comprises approx 146/147 bp (base pairs) of DNA wrapped around an octamer of small basic proteins called histones; this structure causes negative supercoiling
- Each chromosome is made up of DNA tightly coiled many times around proteins called histones that support its structure (chromosomes are tightly packed chromatin)
What is a promoter region and recognition sites/motifs?
- promoter: DNA sequences that define where transcription of a gene begins (GTF help position RNA pol II here)
- recognition sites/motifs: upstream from promoter region and where TF (activators) bind to (specific motifs for specific TF)
Where does histone modification occur?
– Histone modification occurs at lysine amino acids; involves the addition and removal of acetyl or methyl groups.
What is the process of DNA transcription?
- Sequence specific transcription factors interact with specific DNA sequences (motifs) in response to cell signals
- TF activate co-activators (such as HAT) to bind to it -these co-activators can open chromatin structure by the transfer of acetyl groups- and they also recruit nucleosome remodelling complexes such as SWI/SNF (causes the promoter region to open up so that the GTM can bind)
- Co-activators facilitate the assembly of pre-initiation complex composed of general transcription factors (GTFs) and RNA Pol II on the promoter region
What is RNA pol I, RNA pol II, RNA pol III?
– The key enzyme that mediates transcription of most genes to pre-mRNA is RNA polymerase II (Pol II); Moves stepwise along the DNA, unwinding the DNA helix just ahead of the active site for polymerization to expose a new region of the template strand for complementary base-pairing.
Hydrolysis of high-energy bonds provides the energy needed to drive the reaction forward.
– RNA Polymerase-II also produces four snRNAs (small nuclear RNAs) that take part in RNA splicing
– Pol I and III transcribe genes that encode transfer RNAs and ribosomal RNAs
How do hormones serve as transcription factors?
– hormones are lipid soluble so they can slip through the lipid membrane and bind to their receptor within the cell which then forms a hormone-receptor complex that can get through the nuclear pores and bind to the DNA in the nucleus
How does light affect gene expression/phase shift?
- The suprachiasmatic nucleus (SCN) circadian clock is affected by light through the retino-hypothalamic tract (RHT) leading to glutamate (Glu) release
- Glutamate receptor triggering induces various intracellular responses
- Ultimately resulting in gene expression and phase shifts
