Eukaryotic Gene Transcription: Going from DNA to mRNA

DNA Eukaryotic Gene Transcription: Going from DNA to mRNA: The main difference between DNA and mRNA is hydroxylation of the 2’ carbon The other difference lies in the thymine → uracil difference as shown below The DNA is copied into an mRNA template via RNA Polymerase II mRNA processing occurs via 2 phases: 1. Protection: nucleotide sequences on each end of the mRNA transcript are added in order to prevent degradation that can occur outside of the nucleus (5’ cap is a modified G nucleotide and the 3’ polyA tail is a long sequence of adenine nucleotides) 2. Splicing: remove introns from the mRNA transcript which are simply sequences that do not contain any information about how to construct a protein (snipped out by enzymes known as spliceosomes) RNA Interference: a therapy that disrupts the production of harmful proteins by intercepting and incapacitating mRNA transcripts before they make it to the ribosomes Deoxyribonucleic Acid (DNA): Adenine → Thymine (form 2 H-bonds) Guanine → Cytosine (form 3 H-bonds) Double ring structures are known as Single ring structures are known as The human genome has around 6 billion base pairs spread across 46 chromosomes purines pyrimidines Molecular Structure of DNA: The phosphate group is so acidic that when it is placed into a neutral solution, it loses its proton The ribose sugar has a hydrogen group instead of a hydroxyl group (hence deoxyribose) The two purines: adenine and guanine The two pyrimidines: thymine and cytosine Antiparallel Structure of DNA Strands: Using the number of the ribose sugar, you can tell which is the 3’ or 5’ end of the DNA strand (5’ end is the one with the phosphate attached to the 5’ carbon of the ribose sugar and the 3’ is an OH group on the 3’ carbon of the ribose sugar) Two DNA strands are antiparallel because one side runs 3’ → 5’ and the other runs backwards 5’ → 3’ Telomeres & Single Copy DNA vs. Repetitive DNA: Telomeres: present at the end of chromosomes (in eukaryotes because proks have circular DNA) and help protect DNA from damage/degradation They also prevent chromosomes from sticking to one another Repetitive sequence (TTAGGG) Everytime the chromosomes replicate, some of the telomere is lost and it therefore gets shorter Telomerase: able to lengthen telomeres in order replace lost telomere - which is often due to Okazaki fragments. Also, cells that replicate more have a higher concentration of telomerases. Single Copy DNA: holds most of the organism’s important genetic information, these are transcribed/translated, and have a decreased mutation rate Repetitive DNA: found near centromeres, may contain genes that are transcribed/translated, and have an increased rate of mutation Highly Repetitive DNA: contains no genes and is not transcribed/translated Leading & Lagging Strands in DNA Replication: You can only add nucleotides on the 3’ end of the DNA molecule, you cannot add nucleotides to the 5’ end Topoisomerase: allows for the DNA to become unwound ahead of the replication fork in order to allows DNA Helicase to unzip the DNA strands from one another by breaking the hydrogen bonds between the nitrogenous bases DNA Primase: adds an RNA primer to the beginning of the replication strand because DNA polymerase cannot immediately begin copying DNA (for some weird ass reason) Leading Strand: the DNA strand going from 3’ → 5’ since it replicating strand will be 5’ → 3’ allowing for continuous replication Lagging Strand: the DNA strand going from 5’ → 3’ since its replicating strand will be 3’ → 5’ it needs to be done in sections known as Okazaki Fragments (where there are multiple unbound sections of replicated DNA that need to be bound by DNA Ligase - it also removes the RNA primer which was placed there originally) Transcription & mRNA Processing: Remember, in euks transcription occurs in the nucleus (pre-mRNA that still needs to be processed before leaving the nucleus and being translated) while in proks it can occur as the mRNA strand is still being transcribed Promotor: a DNA sequence that tells the RNA polymerase where to attach (the TATA Box can be found in this area as a noncoding DNA sequence) RNA Polymerase: is able to separate the DNA strands and begin copying the information into an mRNA template (can also only make transcripts in the 5’ → 3’ end, which allows for continuous transcription and no fragments like we had in DNA replication) The template strand is the one being copied for the transcript (3’ → 5’) and the coding strand is the exact code that is on the mRNA transcript (only difference is that you have uracil in the mRNA and thymine in the coding strand) Terminator: signals the RNA polymerase to stop transcribing and there are multiple ways to do this: Can form a hairpin structure in the mRNA transcript to stop any further transcription Can be certain DNA sequences that cause a conformational change in the RNA polymerase that makes it let go of the DNA strand Remember that the pre-mRNA strand needs to be processed by splicing out any introns, introducing a modified Guanine to the 5’ cap (helps recognize ribosomes for translation) and a poly-AAA tail to the 3’ end of the mRNA sequence A mature mRNA only has exons, a 5’ cap, and a poly-AAA tail which can then be transported out of the nucleus and into the cytoplasm for translation Speed & Precision of DNA Replication: DNA polymerase is shown to go as fast as 700+ base pairs/second (can speed up or slow down, but on average) Even though it is a fast process, there aren’t very many mistakes on the part of the DNA polymerase (only about 1 mistake/107 nucleotides) Once proofreading occurs, this error rate decreases all the way down to 1 mistake/109 nucleotides Translation (mRNA to Protein): Ribosome: made up of protein and ribosomal RNA (rRNA), it also reads mRNA from the 5’ → 3’ Hace 3 separate sites, A-site (where the tRNA originally binds and amino acid is added to the sequence), P-site (where the polypeptide chain is actually forming), and E-site (where the tRNA exits the ribosome) In euks it is made up of a 60s and a 40s subunit (80s total) In proks it is made up of a 50s and a 30s subunit (70s total) tRNA brings in the anticodon to the mRNA codon (image shown on the right → ) The bottom portion contains the anticodon and the yellow section at the top contains the amino acid that it is carrying to the ribosome Stop codons are UAA, UAG, and UGA (u are awesome, u are gone, and u go away) 61 codons code for amino acids and the 3 above code for stop Differences in Translation Between Proks & Euks: Prok Translation: has a non-coding region (not going to be read by the ribosome), following by the Shine-Dalgarno Sequence (this is where the ribosome recognizes and binds to the mRNA), followed by another non-coding region, and then the start codon (still AUG). The ribosome will read until it reaches a stop codon, and a final non-coding region will be left that is not read as well. Euk Translation: ribosome will bind to the methylated guanine (5’ cap), followed by a non-coding region, and then the ribosome hits the start codon and translates until it hits the stop codon (and finally a last non-coding region). Finally, how come proks don’t have a poly-AAA tail? Well it's because translation and transcription occur in the same place/time and therefore there isn’t even enough time for the mRNA strand to even be degraded like there is in eukaryotic mRNA Last difference is that in proks the first amino acid is always formyl-methionine (methionine with a formyl group attached to it) and in euks it is simply methionine In the human body, formyl-methionine acts as an alarm system because it tells the body that there are likely bacteria present and it will trigger an immune response DNA Repair 1: DNA Polymerase III: synthesizes new DNA and it also has the ability to proofread (make sure that the DNA it is making is correct) This means that if it senses an error, that the DNA pol will actually go backward and remove the incorrect nitrogenous base and replace it with the correct one (known as exonuclease activity which is 3’ → 5’) but can only do this on the end of the DNA strand DNA Polymerase I: removes the RNA primer at the end of replication (exonuclease activity in the 5’ → 3’ direction) and has proofreading capabilities as well Mismatch Repair Mechanism: composed of proteins that first recognize a problem in the sequence of DNA (since mistakes tend to distort the sugar backbone of the DNA), then they mark the DNA with a cut (cuts around the incorrect base), and an exonuclease removes the incorrect nucleotide. A DNA polymerase will then insert the correct nucleotide and DNA ligase will come in and connect the new nucleotide to the nucleotide on its sides. In proks, we know that the mismatch repair mechanism knows that incorrect sequence is on the newly synthesized DNA strand because the original one has methylated adenines all throughout the sequence DNA Repair 2: Remember, a mutation is NOT the same as DNA damage A number of things can cause damage to DNA, such as exogenous or endogenous factors Exogenous Factors: 1. Ultraviolet (UV) Light: can cause the formation of pyrimidine dimers (can be fixed through a process known as nucleotide excision repair (an endonuclease removes the pyrimidine dimer or any nucleotide that isn’t properly arranged, DNA polymerase comes in and places correct DNA bases, and DNA ligase comes in and reconnects the backbone) 2. Gamma Rays 3. X-rays Endogenous Factors: Reactive Oxygen Species: such as a superoxide anion (O2- → oxygen molecules with a free radical) or even peroxides which are molecules that has 2 oxygens and 2 R groups on the outside (ROOR | such as H2O2) → antioxidants can protect against these peroxides If a cell has a lot of damaged DNA, 3 main things can happen to the cell: 1. Goes into a dormant state known as senescence where it doesn’t really do anything anymore 2. Goes into apoptosis which is programmed cell death 3. Goes into unregulated cell division which can cause cancer Semiconservative Replication: In biology, there is a semi-conservative way of replicating DNA since one strand is the original in each of the replicated strands Meselson-Stahl Experiment proved this semi-conservaitve mechanism of replication Protein Modifications: Once the protein is synthesized, it will begin folding into its final 3 dimensional structure with the aid of other proteins as well There are 2 main types of protein modifications: 1. Co-translational Modification: modifications to the polypeptide while translation is occurring a. Acetylation: methionine is removed and replaced with an acetyl group 2. Post-translational Modification: most fall under this category and happen after translation (most happen in the RER and the golgi) a. Glycosylation: the addition of a carbohydrate to a protein (typically to proteins that end up embedded in the cell membrane which helps identification of specific types of cells - such as in the case of red blood cells) b. Lipidation: when a lipid is added to a protein that will be attached to the cell membrane (helps tether it to the cell membrane) c. Phosphorylation: when a phosphate group is added to a protein/enzyme causing a conformational change in the protein (such as in the case of the sodium-potassium pump) and doesn’t typically occur in the RER or golgi, it typically occurs while it is doing its job d. Methylation: if histones are methylated, then the genes are turned “off” e. Proteolysis: the protein needs to be cut in order to be activated (such as in the case of a zymogen) f. Ubiquitination: the addition of a ubiquitin molecule to the protein which marks the protein to breakdown Jacob-Monod lac operon: The only difference between different cells are which specific genes are active within it since all cells have the same DNA present Also, usually gene expression will be regulated at the level of transcription The lac operon is found in the bacteria E. coli (prok) and have a couple different sections (note: glucose is normally used as the energy source for E. coli but when not present, the lac operon becomes important): Structural Genes: codes for lac Z (beta-galactosidase which breaks lactose into glucose and galactose), lac Y (lactose permease which helps bring lactose into the cell), and lac A (not as important for understanding lac operon function) Start Site: located right before the structural genes Stop Site: located right after the structural genes and is where RNA polymerase stops transcribing Promoter Site: RNA polymerase just sits there Operator Site: right after the promoter site and is where the repressor binds (keeps the RNA polymerase from transcribing the genes) When lactose levels are high, a lactose molecule will bind to the repressor and remove it from the operator site, allowing transcription of the genes that break down lactose