The big factorythe book of life

Dr Nisreen Tashkandy

King Abdulaziz UnivesitySeptember 2019

Food process in a factory

• Orders are send to the factory, saying we need a certain product due to high market demand.

• Workers receive the order.

• Send signals to other workers to open the recipe page.

• Follow the instructions of the recipe( template ).

• Cook the raw ingredients under certain temperatures.

• Send on a special route while checking quality control management .

• Preparing packages.

All of above requires a source of power, clean areas , well organized systems and professional employees etc…

Inside the nucleus of every cell in our bodies

• a drawer is like your chromosome in a file cabinet, when your cells need information from the files.

• that particular drawer takes out the file which represent the Gene and make a copy of the information (the RNA molecule) which can travel out into the world (the cytoplasm).

• The genetic factories are the heart of your body’s system of making all the chemicals the body needs and as any factory it needs the place, power, raw material and professional diverse workers.

• you've got six billion, of which 3 billion come from your mother and 3 come from your father.

• Those two copies are attractively similar, so every cell has a genome of three billion, it has two copies of it.

• Your genes are valuables so it needs some sort of protection, that’s why are kept safe in the nucleus and it gives a copy of it self without damaging the original DNA.

• DNA that is transmitted to daughter cells must be accurately duplicated to maintain genetic integrity and to promote genetic continuity.

• If one cell is going to divide to produce two new cells, the first cell must copy all of its parts before it can split in half and copies its genetic information in a process called DNA replication.

The basic steps of DNA replication

• 1- the two parental DNA strands separate , only part of the original DNA strand opens up at one time.

• The partly open/partly closed area where the replication is actively happening is called the replication fork.

• 2- the enzyme DNA polymerase reads the DNA code on the parental strands following the base pairing rules and build new partner strands that are complementary to the original strands .

• 3- its considered semiconservative because each new DNA molecule is half old and half new.

Antiparallel strands

• The parental strands of the DNA are oriented to each other in opposite polarity as the chemicals at the end of each strand of DNA are different from each other

• The 5`end of one strand lines up with the 3` end of the other strand

• The two DNA strands have to be flipped relative to each other in order for the bases to fit together

• There is one problem for DNA polymerase , is that its one way enzyme

Leading vs lagging strands

• The leading strand grows in a continuous piece , the 3`end of this new strand points towards the replication fork with a primer made from primase stuck at the 3` end 5` to 3`primer

• The lagging strand grows in fragments, as the 5` end points away from the fork with primer 5` to 3` in the opposite direction called Okazaki fragments

These fragments need to be connected with ligase which does the ligation

The helper enzymes

• 1- Helicase: separates the original parental strands to open the DNA.

• 2-Primase puts down short pieces of RNA called primers.

• 3-DNA polymerase III removes the RNA primers and replaces them with DNA.

• 4-DNA ligase forms covalent bonds in the backbone of the new DNA molecules to seal up the small breaks created by the starting and stopping of new strands

So lets look at the replication’s recipe

• Template strand of DNA

• floating around in the solution some free nucleotide

• It needs a head start as primers at the 5` end and the other end is 3` end hydroxyl group

• The helper enzymes

• the DNA polymerase gets energy for adding triphosphate in a certain location on the DNA strand and joining it in a sugar phosphate chain

Hydrolysis of the ATP

• Adenosine triphosphate is comprised of adenosine bound to three phosphate groups

• Adenosine is consisting of the nitrogenous base adenine and a five-carbon sugar, ribose.

• The three phosphate groups, in order of closest to furthest from the ribose sugar, are labeled alpha, beta, and gamma.

• Together, these chemical groups constitute an energy powerhouse. However, not all bonds within this molecule exist in a particularly high-energy state.

• Both bonds that link the phosphates are equally high-energy bonds (phosphoanhydride bonds) when broken, release sufficient energy to power a variety of cellular reactions and processes.

Topological characteristics of DNA

DNA supercoiling influence all major DNA transactions in living cells.

DNA supercoiling induces the formation of unusual secondary structure by specific DNA repeats which can also affect DNA functioning.

Weather DNA entangled or separated like strands it remain chemically the same but topological different ( how they are wrapped ).

Topoisomerase II enzyme cut the these areas.

Fidelity accuracy of replication –equilibrium

The fidelity of DNA replication relies on nucleotide selectivity of

1- replicative DNA polymerase Replication = 10 -3

2- exonucleolytic proofreading Proof reading = 10 -6

3-post-replicative DNA mismatch repair (MMR) : it cuts, removes incorrectly incorporated nucleotides from the primer terminus and gives the cell another chance to do it again MMR = 10 -9

It is estimated that proofreading improves the fidelity by a 2–3 orders of magnitude.

Colon Cancers

• Defects in the DNA mismatch repair (MMR) proteins, result in a phenotype called microsatellite instability (MSI), occurring in up to 15% of sporadic colorectal cancers.

• Approximately one quarter of colon cancers with deficient MMR (dMMR) develop as a result of an inherited predisposition Lynch syndrome

The Big Cookbook

• The Big Cookbook is divided into 46 Sections. (chromosomes ) and the book has an index to find the right page of the chemical to make.

• Each Section is divided into chapters arrangement is called the chromatin code which is a blend of DNA and support proteins called histones.

• Each Chapter has thousands of Pages. (genes)

• Each Page has instructions, and most of them have a template or model of a chemical.

Histone code or Epigenetics

• Each nucleosome is an octamer of eight histones, two of each H2A, H2B, H3, and H4.

• The nucleosomes are wrapped in a spiral structure called a solenoid, and additional H1 proteins are associated with each nucleosome as linkers to maintain the overall chromatin structure.

• There are two states of chromatin; euchromatin is open and amenable to transcription, whereas heterochromatin is a compact DNA-protein structure that cannot be transcribed.

• Chemical modifications to histones cause conversion of DNA from a euchromatin state to a heterochromatin state and vice versa.

Strahl and Allis

coined the term histone code to describe the concept that specific histone modifications could act in combination to form a recognizable “code” that could regulate transcription as well as the state of chromatin condensation.

workshops

• The Big Book – (the DNA) which contains the plans and instructions.

• The template (the RNA).

• Shipping cargo most proteins, ribosomal subunits, and some DNAs are transported through the pore complexes in a process mediated by a family of transport factors known as karyopherins or Importins which mediate movement into the nucleus

whereas Exportins mediate movement out of the nucleus

• Dynamic interplay – (like Cajal bodies) which are regions within the nucleus that are enriched in proteins and RNAs involved in mRNA processing.

Cajal bodies the main sites for the assembly of small nuclear ribonucleoproteins (snRNPs).

• The bath for swimmers – (the nucleoplasm or karyolymph) it’s a viscous liquid This is where everything comes together to make the chemicals.

• work in progress : chromatin fibrils which is playing a role as early markers of transcriptional alterations and for the cellular metabolic state.

Change it from one language to another

• One gene = one blueprint for a functional molecule

• If the cells in your pancreas couldn’t make the protein insulin, you end up with diabetes

• Transcription in cells takes the information in DNA and uses it to make RNA.

• Translation in cells takes the information in mRNA and uses it to build up a protein.

Rewriting DNA message transcription

• The chain that runs along the outside is made of sugar molecules and negatively charged phosphate molecules.

• A,T,C and G are called the nucleotide. Then it goes: sugar/phosphate/sugar/phosphate in a repetitive structure

• 1- RNA polymerase locates the gene for that protein and makes an RNA copy of it

• 2- RNA polymerase slides along the gene trying to match DNA nucleotides in the gene

• 3- the base pairing rules are almost the same with one exceptional uracil ( U)

Transcription factors

• Are proteins possessing domains that bind to the DNA of promoter or enhancer regions of specific genes.

• They also possess a domain that interacts with RNA polymerase II or other transcription factors and consequently regulates the amount of messenger RNA (mRNA) produced by the gene.

• RNA locates the genes it needs to copy with the help of transcription factors that mark the beginning of genes.

Promoters• Core promoters are diverse and may contain a variety of sequence elements such

as the TATA box: located 15 to 30 bp upstream.

• the Initiator sequence (INR) near the RNA start site +1.

• the downstream promoter element (DPE) recognized by the TATA-binding protein (TBP) and TBP-associated factors of the TFIID complex.

• Gene-specific activation by enhancers involves their communication with the basal RNA polymerase II transcription machinery at the core promoter.

Transcriptional terminators

• The ends of genes are marked by a special sequence called the transcription terminators which can work in different ways.

• Polymerase I is responsible for ribosomal RNA.

• Polymerase II is responsible for mRNA and miRNAs.

Polymerases I and II employ different mechanisms to terminate transcription.

• Polymerase III transcribes tRNA and other short RNAs.

Polymerase III relies on a specific sequence and RNA secondary structure to induce transcript cleavage.

Transcription process

• 1- RNA polymerase BINDS to the promoter with the help of transcriptional factors

• 2-RNA polymerase separates the two strands of the DNA double helix in a small area and use one of the strands as a patterns

• 3- RNA polymerase uses base pairing rules to build an RNA strand that’s complementary to the DNA in the template strands

• 4- RNA polymerase reaches the termination sequence and releases the DNA

RNA processing

• After RNA polymerase transcribes one of your genes and produces a molecule of

Primary transcript (Pre-mRNA) 5% because its not quite finished

It has to undergo a few finishing touches via RNA:-

1-A protective cap is added to the beginning of the mRNA to tell the cell it should translate this piece of RNA

2-The poly A tail which is a chain of repeating nucleotides that contain adenine A is added to the end of the mRNA to protect it from being broken down by the cell

3-the pre-mRNA is spliced to remove introns

The Human Ribosomal Protein Genes

• The ribosome, are a catalyst for protein synthesis.

• mammalian cells contain approximately 4 × 106 cytoplasmic ribosomes

• rRNAs, which are encoded by several hundred copies of genes

• each mammalian ribosomal proteins is typically encoded by a single gene.

• Single functional genes generate large numbers of processed pseudogenes What isthe potential benefit to retain pseudogene? There is evidence that interaction ofpseudogenes with their functional genes regulates different biochemical processesin cells.

The eukaryotic ribosome

• is composed of four RNA molecules and 80 ribosomal proteins assembled through a complex series of steps requiring the participation of nearly 300 RNA and protein cofactors.

• Ribosomal mRNA.

• Small non coding RNA (ncRNA) like rRNA, tRNA which are transcribed but not translated.

• Nucleous (snoRNA), nucleus RNA (snRNA) and cytoplasm (miRNA)

The eukaryotic ribosome

• In eukaryotic cells, up to 80% of RNA synthesis belongs to rRNA transcription crucial to the preservation of ribosome biogenesis and protein synthesis. .

• the nuclear rRNA encoded by ribosomal DNA (rDNA), or by rRNA genes.

• There are about 1.5–3 million ribosomes per eukaryotic cell .

• ribosome biogenesis consumes a tremendous amount of cellular energy and rRNA synthesis is tightly linked to cell growth and proliferation, and as such, it is responsive to general metabolism and environmental challenges .

The eukaryotic ribosome

• In Eukaryotes, rRNA genes consist of several distinct multigene families tandemly arrayed as repeats composed of tens to hundreds or even thousands of copies.

• two mitochondrial rRNAs, example:- the 12S.

• the nuclear rDNAs is consisting of a large, nucleolus-forming 45/47S rDNA unit

• a substantially smaller extra-nucleolar 5S rDNA .

• Both the 45S and 5S rDNAs are organized into clusters of repeats often enabling their cytogenetic visualization on chromosomes

key words -

• CP—core promoter.

• ETS—external transcribed spacer, ICR—internal control region, IE—internal element, IGS—intergenic Spacer.

• ITS1, ITS2—internal transcribed spacer 1 and 2, RNA Pol I and III—RNA polymerase I.

• LSU—large (ribosomal) subunit.

• nt—nucleotides.

• NTS—non-transcribed spacer.

• SSU—small subunit.

• TIS—transcription initiation site.

• TTTT—poly T transcription termination site.

• UCE—upstream.

MicroRNA

• miRNA is encoded by genes and it’s a single stranded RNA molecules about 21 to 23 nucleotide in length.

• Their function is regulating gene expression by their ability to bind mRNA.

GC Content of rDNA NCBI, human genome assembly• The 45S rDNA gene clusters form the GC-richest genomic fraction particularly in

Eukaryotes.

• with humans having 60%–80% GC in different parts of the rDNA.

• The GC-richness is due to the recombination rate based process known as GC-biased gene conversion.

Three different RNA polymerases are involved in production of these RNAs and proteins

• RNA polymerase I (POL I) is involved in production of the 28S, 18S, and 5.8S rRNAs.

• POL II in production of ribosomal proteins. mRNA, microRNA.

• POL III in production of the 5S rRNA.

• The amino acid sequences of all rat and human ribosomal proteins have been presumed.

• The nucleotide sequences of thousands of eukaryotic rRNAs are now known (The Ribosome Database Project).

The Genetic Codes universal amongst all of life

• AUG start codon which represent the methionine

• UAG, UAA and UGA called the stop codon

• All three nucleotides are called triplets

• How many three letter words are there with four nucleotide? 64

• How many amino aside are there? Redundancy

• Arginine is represented by more than CGU,CGC,CGA and CGG

Translation help workers • Transfer ribonucleic acid (tRNA) is a type of RNA molecule that helps decode a

messenger RNA (mRNA) sequence into a protein.

• One of these hairpin loops contains a sequence called the anticodon, which can recognize and decode an mRNA codon.

• Each tRNA has its corresponding amino acid attached to its end.

• When a tRNA recognizes and binds to its corresponding codon in the ribosome, the tRNA transfers the appropriate amino acid to the end of the growing amino acid chain.

Reading the book of life

• After mRNA leaves the nucleus of a cell it heads for a ribosome in the cytoplasm

• The strand slides through the ribosome

• The code is read three nucleotide at a time

Initiation

• the small subunit of the ribosome binds to the mRNA

• The first tRNA molecules which carries the amino acids attaches to the start codon

• The start codon is AUG complementary to UAC anticodon on the tRNA

• The large subunit of the ribosome attaches to form a complete ribosomes

• There are three pockets on the complete ribosome, the A site , the P site and the E site

• The tRNA enter the ribosome at the A site then move to the P site then finally exit through the E site

Elongation

• The tRNA enters the A site

• The adjacent P site holds a tRNA with the growing polypeptide chain

• If Two tRNA are parker next to each other in the A site and the p site the ribosome catalyzes the formation of a peptide bond between the growing polypeptide chain

Termination

when stop codon enters the A site

An enzyme called a release factor enters the ribosomes and cuts the polypeptide chain free

Class I release factors: Class I RF, eRF1, that can recognize all three stop codons

Class II release factors are GTP-binding proteins that serve to enhance the efficiency of termination, either by promoting release of the Class I factor from the ribosome after peptidyl release or by forming a complex with the Class I RF prior to stop codon recognition

Translation stop

Ribosomes and mRNA separate from each other

The final product• Polypeptide chains may be modified before they fold up and become functional

proteins

• Often more than one polypeptide chain combines with another chain to form the complete protein