Appunti di Genetics

RNA Polymerase in Eukaryotes

RNA polymerase in eukaryotes consists of five different types.

• Type I: Responsible for transcribing rRNA (28S, 18S, 5.8S)
• Type II: Responsible for transcribing mRNA, snRNA, and miRNA
• Type III: Responsible for transcribing tRNA, rRNA (5S), and some siRNA
• Type IV: Specifically involved in transcribing siRNA in plants
• Type V: Involved in siRNA-directed heterochromatin formation in plants

In addition to RNA polymerases, there are RNA-dependent RNA polymerases (RDRPs) that play a crucial role in gene silencing after transcription.

RNA polymerases are composed of multiple subunits. In yeast, for example, there are 11 subunits forming a multiprotein complex, some of which are similar to those found in bacteria.

Eukaryotic promoters contain various elements, including:

• The TATA box, which is frequently found but not always present, typically located around -25
• The CAAT box
• The GC box, located between -50 and -200

While none of these elements are essential on their own, together they contribute to accurate transcription.

DNA transcription in eukaryotes is complex. Enhancer sequences and silencers are present in the DNA, to which activators and repressors (proteins) bind. Some proteins recognize specific sequences and bind to them.

repress it directly but justinteract and brings with it a repressor. But this effect can change with a different environment or internal signal: theprotein can bring with it an activator if there’s a signal to do so. So often enhancers and silencers are the samesequence and it all depends on what protein its binding protein brings with it. The activators stimulate thepolymerase. The structure is not linear – promoter complexes can be physically very distant but in the nucleus theyare close thanks to loops. There many cofactors involved in the whole process.

The chromatin structure is important and there are 3 levels of regulation of the polymerase: histone variants that area little bit changed from the normal ones which gives a different packaging in the DNA. There is also acetylating(modification) and methylation; and positioning (the promoter can in the nucleosome, so factors cannot bind it).

An mRNA has a 5’ UTR (untranslated region), initiation codon, ORF,

termination codon, 3’ UTR.5’ capping: the cap is a 7 methylguanosine added by guanyltransferase and this modification is essential for translation in all eukaryotes. It also stimulates splicing and poly A addition. The 2 step is that you can have a poly A tail which is not encoded by the DNA sequence but it’s added later. There is a specific site (cleavage site) in the RNA and an endonuclease cuts the cleavage site and adds poly A strand which is very important for the stability of the RNA and for the function of the mRNA.

The 3 step - split genes: eukaryotic genes are interrupted by introns (interrupting sequences) which are removed by splicing. So, there is a maturation of the RNA into an ORF. Some eukaryotic genes don’t have introns and some have giant introns. The coding part are the exons. The DNA is much longer than the RNA and the loops are the introns which have been removed (DNA-mRNA duplex). If you compare the RNA sequence with the DNA sequence, the

Parts that are missing are the introns which have been spliced out.

Prokaryotes vs eukaryotes: no introns, no capping, no poly A, much simpler, translation and transcription at the same time (linkage between them). Eukaryotes have nucleus, capping, splicing, poly A, translation in the cytoplasm.

The splicing is a regulatory process (a gene can be spliced in different ways to get different proteins).

NO exam: There are small RNAs (21-24 nucleotides) which are involved in gene silencing (RNA interference – gene silencing by double-stranded RNA) and they do that by complimenting to the poly A tail of an mRNA – it can cut it or inhibit the translation (transcription silencing). Small RNAs can also modify the chromatin and make it heterochromatin (post transcription silencing). Dicer and Argonaute are involved. All this process is based on the formation of a double stranded RNA. If the cell sees double stranded RNA, Dicer chops it in small pieces while the Argonaute is important for

Finding this small RNA and binding it and silencing it. There are 2 pathways: small interfering RNAs (siRNA) and microRNAs. siRNA is based on double stranded RNA, Dicer cuts it and Argonaute confers silencing or inhibiting its function by binding it directly. It's a defence mechanism and these interfering RNAs are produced by invading viruses, transposons, aberrant genes. MicroRNA (regulatory RNAs that control the expression of genes) is encoded by specific genes and they come in double stranded RNA. These are processed by Dicer and one of the 2 strands is used by Argonaute to complement to the mRNA and it can cut it or inhibit the translation. A gene can be expressed everywhere (the promoter is everywhere) and microRNA are expressed everywhere but one place, and it's there where the gene will become protein (in other places it will be degraded). So it regulates genes' activity: where the gene is expressed – no miRNA. Where it's not expressed – miRNA.

degrades it.

How genes work:

Garrod in 1902 got first clues on what were the functions of genes from studies of humans and he was the first to prove a relation between genes and enzymes. He studied Alkaptonuria which is a disease by which the urine turns black when exposed to air (oxidation). It’s an early post mendelian period: based on pedigree analysis hereditary defect produced by recessive mutations. In 1908 he proposed that the disorder was caused by the lack of an enzyme that normally splits the aromatic ring from homogentisic acid. This is a metabolic pathway where one of the genes is mutated. And this homogentisic acid is accumulated because the oxidase is not working, so you don’t get the conversion into maleylacetoacetic acid. Metabolic pathways have a substrate and a final product but there are different intermediates, so you need different proteins which are encoded by different genes.

Phenylketonuria is an autosomal recessive disorder which has to do with the lack of

Conversion of phenylalanine into tyrosine occurs due to a deficiency in phenylalanine hydroxylase (PAH). Phenylketonuria (PKU) is one of the few genetic diseases that can be controlled by diet. If left untreated, it can lead to severe brain disorders and irreversible seizures.

The treatment for PKU involves a low phenylalanine diet and a high tyrosine diet. This is because a normal diet would result in the accumulation of phenylpyruvic acid, which is toxic. Since PAH is not active in individuals with PKU, the production of this toxic acid is increased. However, by providing a high tyrosine and low phenylalanine diet, the patient can continue the rest of the metabolic pathway normally.

It is important to note that in a metabolic pathway, each step is catalyzed by a different enzyme encoded by different genes. Therefore, if one step is blocked, providing a substrate downstream of the issue will allow the pathway to continue functioning.

The "one gene - one enzyme" hypothesis was proposed by Beadle and Tatum. They conducted experiments using Neurospora, a fungus with a haploid genome, and produced mutants.

single gene. With X rays there are a lot of mutations, but they didn’t want double mutants. If you have the original ascospore and you get a mutation and cross it with wild type, the cell becomes a 2n (diploid) which is heterozygous. Then you get duplication (4n) and then meiosis and get 4 and then 8 ascospores and see a 1:1 segregation ratio, so the conidia you get will segregate into 1:1. If the mutation is made by 2 genes, there is a double mutant crossed with wild type and get a 4n and then meiosis and get a 3:1 segregation (mutants x wild type phenotypes). That’s why they chose single mutants, because if they got 3:1, it could have been 2 mutations. How they did the experiment: took hundreds of tubes of complete media with single ascospores and each is in the single rich medium (complete medium), then they all grow because this media has everything, then they transferred them to minimal medium and those that don’t grow are the ones that interested them because that

Conversion of the single step. So, each gene encoded a different enzyme. Which was which? Instead of giving arginine, they started giving the mutants different substrates. So, in the minimum medium only the wild type grew. Then you can add ornithine and argE starts to grow and the others don't, you can add citrulline argE and argF grew and etc. So, the one that can grow if you add ornithine is argE. So argE encodes for the first enzyme. Then comes the argF, argG and argH. That's how they demonstrated that one gene encoded for one enzyme.

So, in metabolic pathways you have a component A and want to get a component B so if you have an interruption/mutation in gene B, you don't get the enzyme beta so you have to give the substrate that's after the mutation. So, you need to get component C. So, if you have a mutation in gene B, B will accumulate and maybe part of it will turn back into A and if A is part of another pathway, there's deregulation in other things also.

For every auxotroph mutant there is a selective medium (you have to see what it needs). For example, E. coli: it's a bacterium that divides by binary fission (the cell grows, comes to a limit, and goes in mitosis and you get 2 bacteria). You can get a colony from a single bacterium. They grow on agar plates.