3.1.3 Regulation of gene expression and 3.1.4 Transcription, RNA processing and translation Flashcards
Where do transcription and splicing occur in eukaryotes?
In the nucleus (compartmentalisation)
Where does translation occur in eukaryotes?
In the cytoplasm (compartmentalisation)
Do prokaryotes show compartmentalisation during gene expression?
No, all three processes occur in the cytoplasm (no membrane-bound organelles).
What proteins carry out transcription?
RNA polymerases (four types in eukaryotes, one type in prokaryotes)
What organelle is involved in translation?
Ribosomes
What monomer is used to form RNA?
Ribonucleoside triphosphates
What do RNA polymerase enzymes use to form the RNA transcript?
They use one of the strands of DNA as a template (the strands are separated by a helicase component of the enzyme itself), and the other strand is known as the ‘coding strand’ as the transcript will have a similar structure (thymine, T, bases replaced by uracil, U, bases) to that strand.
In which direction do RNA polymerases form transcripts?
5’ to 3’ (so add on to the 3’ end), same as DNA polymerases - therefore moves along the template strand in a 3’ to 5’ direction. Either strand can be transcribed, depending on the direction/orientation of the polymerase enzyme.
What is the main difference between DNA and RNA polymerase enzymes?
DNA polymerases require a primer, RNA polymerases do not.
What are the products of RNA polymerase I?
rRNAs (ribosomal RNAs)
What are the products of RNA polymerase II?
mRNAs (messenger RNAs), snRNAs (small nuclear RNAs), snoRNAs (small nucleolar RNAs) and miRNAs (micro RNAs)
What are the products of RNA polymerase III?
tRNAs (transfer RNAs) and some snRNAs (small nuclear RNAs)
What are the products of RNA polymerase IV/POLMRT?
Mitochondrial RNA polymerases transcribe mtRNA - acts upon mitochondrial DNA and transcribes the products there.
What is the function of mRNA?
To act as a messenger and code for proteins
What is the function of rRNA?
To form the core of ribosomes and catalyse protein synthesis
What is the function of tRNA?
To serve as an adaptor between mRNA and amino acids during synthesis - is bound by an ester bond to an amino acid molecule.
What is the function of aminoacyl-tRNA synthetase/tRNA ligase?
This enzyme causes ATP to bind to the amino acid, losing two phosphate groups in the process to form amino acid-AMP. The enzyme then facilitates the transfer of this aminoacyl group to the tRNA molecule, as the AMP is swapped for the tRNA and an ester bond formed. This results in the amino acid to be attached to the tRNA molecule.
What is the function of miRNAs?
These regulate gene expression as part of RNA interference mechanisms - after associating with a RISC factor (RNA-induced silencing complex), these small, single stranded RNA molecules are able to bind to complementary codes on mRNA molecules and occasionally, through use of the Argonaute protein, are able to cause the cleavage of the mRNA, leaving it open to degradation by exonucleases. More frequently, however, the miRNA-RISC complex just initiates the association of more of these complexes, causing translation to be prevented and the mRNA molecule to be shipped off to a protein- or P-body, where the molecule is sequestered from ribosomes and stored/eventually degraded.
What is the function of snRNAs?
Primary function is pre-mRNA processing within the nucleus - they are involved in regulating splicing through binding to short conserved motifs between exon regions.
What is the function of snoRNAs?
Located within the nucleolus, some are used in the processing of rRNA but most just act as guides for other cellular RNAs (mostly snRNAs).
What are the bases available in RNA?
Adenine (A), Uracil (U, replaces thymine), Cytosine (C) and Guanine (G)
Complementary pairings: A U, C G
Where do RNA polymerases associate on DNA strands to initiate transcription?
Gene core promoters
What is an example of a promoter region?
TATA boxes are most common
What are the molecules that facilitate the association of RNA polymerase to the promoter region?
Transcription factors (proteins - multiple in eukaryotes, often only one in prokaryotes)
Which genome is more complex, prokaryotic or eukaryotic?
Eukaryotic genomes are more complex
What do eukaryotic RNA polymerases require that prokaryotic RNA polymerases do not?
The presence of multiple transcription factors, i.e. GTFs (general transcription factors) that link the protein scaffold complex formed at regulatory sites to the RNA polymerase enzyme.
Where do transcription factors bind?
Either directly to the promoter region (GTFs only) or in other cis-regulatory (cis = on the same chromosome - and example of a trans-regulatory site would be a gene which codes for a relevant transcription factor on a different chromosome) site just upstream of the promoter region.
What is a repressor?
This is a transcription factor that inhibits the action of RNA polymerases, for example by completely blocking the promoter region, preventing binding.
What is an activator?
This is a transcription factor that encourages the action of RNA polymerases, for example by providing more regions that can form favourable bonds with the enzyme (incr stability) other than just the DNA strand, and by positioning the enzyme so that it is facing in the correct direction for transcription.
What is a co-repressor?
This is another name for repressors but in eukaryotic cells - as there are often multiple transcription factors needed, the nomenclature changes, and these factors do not interact directly with the enzyme.
What is a coactivator?
This is another name for activators but in eukaryotic cells - as there are often multiple transcription factors needed, the nomenclature changes, and these factors do not interact directly with the enzyme.
How do transcription factors/GTFs bind to DNA?
This is due to various regions on the proteins (i.e. hydrophobic areas, electron donating or accepting regions) that recognise specific sequences on the DNA strand - because of this, gene expression is highly regulated as each transcription factor can be specific to a promoter region and an RNA polymerase enzyme. They can also bind cooperatively, forming dimers before function is allowed, and different factors can have different regulatory effects depending on the combination they are in.
Give examples of GTFs/general transcription factors for RNA polymerase II.
TFIIA, TFIIB, TFIID, TFIIF, TFIIE, TFIIH
What is a pre-initiation complex (PIC)?
This is the scaffold of proteins formed at a promoter/regulatory region that control transcription and allow for its initiation.
- Go through the process of GTF association for RNA polymerase II
TBD subunit of TFIID associates with a TATA box
TFIIB joins through recognising and associating with a BRE sequence on the TFIID
TFIIF recruits RNA polymerase II and stabilises its interaction with TFIIB and TBP
TFIIE and TFIIH join (subunits of the TFIIH protein have helicase properties, which is required to unwind sections of DNA)
After this point, the pre-initiation complex has been formed
(Subunits of the TFIIH also have kinase activity which will phosphorylate the C-terminal domain of Pol II, which ends abortive initiation/triggers transcription).
Why are transcription factors useful?
They are able to interact with/be affected by the environment, allowing the cell to have timely and relevant responses to tissue specific and proliferation cues as well as various stimuli.
Which regulatory complexes are more complicated?
Eukaryotic
What are distal regulatory elements?
Regions of DNA that can regulate gene expression but can be up to 1mb away from the promoter region itself
What is DNA looping?
This is where the stands of DNA are looped through the action of proteins to bring regulatory and promoter regions physically closer together. This allows for better interaction between the two regions and proteins associated at either sequence. The method is seen in both prokaryotes and eukaryotes.
What is the function of an enhancer element?
To activate expression of a promoter region over long distances
What is the function of a silencer element?
To repress transcription at promoters
What is the function of an insulator element?
This is used to regulate expression between neighbouring genes/prevents other regulatory sites from affecting genes other than their target.
What is the function of a locus control region?
These regulate the expression of a cluster of genes to ensure correct sequence of expression.
What allows distant sequences to impact upon transcription?
DNA-protein and protein-protein interactions.
What component of eukaryotic DNA allows for more regulation than that of prokaryotes?
Chromatin structure and the presence of nucleosomes/histones.
How can histones affect gene expression?
Due to the opposite charges between histones and DNA (histones contain many basic amino acids, so are positively charged, whereas the backbone of DNA contains many phosphate groups so has a high negative charge), the two have high electrostatic attraction and so associate quite closely.
This can be altered through histone modifications, however.
DNA must actually be accessible for the association of transcription factors to occur - chromatin and histone modifications can affect this.
What is the term for dense chromatin (many nucleosomes)?
Heterochromatin - also associated with additional heterochromatin proteins that bind to the specifically modified histone tails in that region
Genes in heterochromatin are…
Effectively silenced, gene expression is prevented.
What is the term for ‘looser’ chromatin (fewer nucleosomes)?
Euchromatin - can be regulated by histone tail modifications. Histones are positioned further apart.
Genes in euchromatin are…
Able to be transcribed - these regions often contain active genes.
What are the general features of eukaryotic transcription factors?
DNA binding regions
Regions to associate with other regulatory proteins or associate directly with the polymerase enzyme itself (latter is GTFs only)
What are potential histone tail modifications?
Acetylation (addition of an acetyl group, CH3CO)
Methylation (addition of a methyl group, CH3)
Phosphorylation (addition of a phosphate, PO4^3-)
Which terminal do histone tails express?
N-terminal
What can be the effect of histone tail modifications?
A gene can either be silenced (due to the promoter region becoming closely bound to the histone, preventing transcription factors association) or activated (DNA unwound from histone due to modification, allows for binding of transcription factors and initiation of transcription).
What is the affect of acetylation of histones?
By adding negatively charged acetyl groups, DNA dissociates from the histone protein due it being repelled by the like charge/the basic charge of the histones is reduced.
The opposite occurs/DNA associates more closely during deacetylation.
What enzymes carry out acetylation and deacetylation of histones?
HATs carry out acetylation (histone acetyl transferases)
HDACs carry out deacetylation (histone deacetylases)
What is the effect of methylation of histones?
DNA is activated/dissociates from the histones after methyl groups are added, due to the CH3s binding to the basic amino acids that make up the histone protein - this reduces the positive charge of the histone and causes the electrostatic attraction between the two species to be decreased.
The opposite occurs/DNA associates more closely during demethylation.
What enzymes carry out methylation and demethylation of histones?
Lysine- and arginine-specific methyltransferases carry out methylation
Demethylase enzymes remove methyl groups from nucleic acids (especially histones)
What is the effect of methylation of DNA and where does it occur?
This occurs most commonly at CpG sites (regions where cytosine and guanine bases are adjacent) and methylation of DNA prevents the action of RNA polymerase enzymes, so silences genes/prevents transcription.
The opposite occurs/transcription is possible once the methyl groups are removed.
What enzyme maintains methylation of DNA?
This is the Dnmt1 enzyme (DNA methyltransferase 1 enzyme)
What is the effect of phosphorylating histones?
Phosphate groups are highly negative, so will cause dissociation of DNA, but phosphorylation of histones also allows for histone sliding, where the histones - as they are dynamic structures - are able to ‘slide’ up and down the DNA strand, so will therefore affect different regions of DNA.
What enzymes carry out the phosphorylation and de-phosphorylation of histones?
Kinases phosphorylate, phosphatase enzymes remove phosphate groups.
What regions of DNA are likely to be heterochromatic?
Regions of DNA that don’t contain any genes, or that contain inactivated that are not necessary within the cell.
- What is an operon?
This is a series of genes in sequence (more commonly seen in prokaryotes) that all code for the same enzyme.
- How did the study of operons lead to the Jacob-Monod model of gene regulation?
Through observations of the trp and lac operons in bacteria (E.coli), the latter in particular. The Jacob-Monod model suggested that a region of DNA just upstream of the transcription initiation site (promoter region) has the ability to associate with transcription regulators/factors.
In the trp model, the enzyme coded by the operon catalyses the formation of the amino acid tryptophan -however, when the bacteria is in a solution with a high conc of the amino acid, this operon is repressed/the production of the enzyme halted. This is because the amino acid binds to an inactivated repressor protein in the cytoplasm and activates it (x4 molecules needed), meaning it can then bind to its complementary regulatory site and inhibit the action of the prokaryotic RNA polymerase enzyme. Once the extracellular conc of tryptophan decreases, the factor dissociates and action of RNA polymerase is allowed again.
In the lac model, the enzyme coded for by the operon catalyses the digestion of lactose sugars. When the enzyme is in a solution with a high conc of glucose, this enzyme is not needed and so is repressed by the lac repressor protein. The lac repressor protein is an indirect sensor for lactose, becoming inactivated when its isomer, allolactose, detects the disaccharide. Catabolite activator protein (CAP) acts as an activator protein when the concentration of lactose is high and glucose is very low - this protein is able to indirectly sense when the glucose concentrations are low (through cAMP messengers), only then activating the lac operon at an upstream regulatory site.
What is constitutive heterochromatin?
Regions of heterochromatin that mostly contain a high number of tandem repeats (satellite DNA), i.e. centromeres and telomeres are permanently heterochromatic.
- What enables X inactivation in mammals?
The self-propagating nature of heterochromatin (can also be problematic when regions of heterochromatin undergo translocations into regions of euchromatin).
In particular, the expression of the Xist gene on both X chromosomes covers regions of DNA, causing it to be silenced.
- What is the result of X inactivation?
Regions of the X chromosome are permanently silenced - this is important for gene regulation as if all of the same genes on both of the homologous chromosomes were expressed there would be too many proteins within the cell, so one of the genes must be silenced (different genes between the two chromosomes are silenced, however, not always just one chromosomes becoming completely inactivated).
What is a higher order chromatin structure that can affect gene expression?
To silence large stretches of DNA, chromatin can associate/bind to the nuclear lamina through associations with proteins bound to the membrane.
How can gene expression be regulated by external messengers?
Hormones and other messengers can either diffuse into the cell (i.e. oestrogen) or bind to receptors on the cell surface, causing the activation of various transcription factors to activate or repress genes.
