Molecular Genetics Lec (1) PDF

LESSON # 1: Central Dogma of Molecular Biology Trans Checker: Estrella Trans Makers: Estrella Completed Alfonso Completed Fenis Completed ____________________________________________________________________________ 0:00-40:00: Estrella What is a dogma? Dogma is a set of principles...

LESSON # 1: Central Dogma of Molecular Biology Trans Checker: Estrella Trans Makers: Estrella Completed Alfonso Completed Fenis Completed ____________________________________________________________________________ 0:00-40:00: Estrella What is a dogma? Dogma is a set of principles and beliefs that is not debatable. It’s not a rule, it’s a set of ideas and concepts that are universally agreed upon as the ultimate truth. Central Dogma of Molecular Biology is the central concept of molecular biology that is the absolute truth. Why wasn’t it called the Law of Molecular Biology instead? Central Dogma It is a conceptual framework in which it posits that the flow of genetic information is only one way, which is from DNA to RNA and then ultimately to protein, which then provides various cellular functions such as for structure and cellular metabolism. There are two forms of molecules where genetic information is stored: ○ DNA (deoxyribonucleic acid) ○ RNA (ribonucleic acid) Difference between DNA and RNA  LESSON # 1: Central Dogma of Molecular Biology All somatic cells in our body contain all information of all genes or instructions. DNA is the main storage molecule of all the genetic information to make us and all somatic cells contain the same DNA. For example, the cells in your hands contain also genes that have the information to make other cells. Then, how does your cells know that the skin cells that they will create is for the face or if it is for the other parts of your body? Or why doesn’t the heart produce bile? How can these cells know their function? If every cell has a blueprint, why do we have different types of cells? ○ This is because cells only express the genes that they need and not the whole bunch. ○ But, how can we measure genetic expression? Through the presence of mRNA. ANALOGY: Suppose DNA is this book of life or this very long blueprint. Then, a cell asks how to make a liver cell. Is it efficient to give the whole blueprint even if the cell is just asking how to make one type of cell? Or would it be more efficient to just give it a usable copy of just the desired part? DNA (book) -> RNA (usable copy)  LESSON # 1: Central Dogma of Molecular Biology However, it doesn’t mean that we don’t need DNA, it is needed because it is the grand repository of all the information about us. The Central Dogma is designed like that. From DNA, we get the necessary information which is the RNA. This instruction is used to make a functional protein. DNAs and RNAs are linear polymers composed of repeating units or monomers of a molecule called nucleotides. Nitrogenous Bases: Adenine (A)  LESSON # 1: Central Dogma of Molecular Biology Guanine (G) Thymine (T) Cytosine (C) Uracil (U) These nucleotides are always complementary to one another. Adenine will always pair with Thymine or Uracil, while Cytosine will always pair with Guanine. Question (True or False): The individual nucleotides contain the information and instructions on how to build protein themselves. Answer: False. Because the instructions on how to build proteins is in the order of nucleotides, not the letters themselves. For example: ○ Olcruaelm nitegces will not give you any information. A word not arranged does not hold any information. DNA is deoxyfied or has missing oxygen while RNA has. In the 2’-end of the ribose sugar in RNA, there is a hydroxyl group (OH) while in the deoxyribose, there is only a proton. This differentiates them when it comes to stability. ○ RNA is much more prone to degradation in some conditions. For example, in a basic environment, these hydroxyl groups can react with the adjacent phosphate group, which is negatively charged.  LESSON # 1: Central Dogma of Molecular Biology This can lead to hydrolysis which can cause the breakage of the RNA strand. ○ That’s why in the laboratory, there is extra precaution in handling RNA. ○ (CHATGPT): The hydroxyl group in RNA makes it more chemically reactive and less stable than DNA. Specifically, this -OH group can participate in intramolecular reactions, such as hydrolysis, where it can attack the phosphodiester bond between nucleotides, leading to the cleavage of the RNA strand. This is one reason why RNA is more prone to degradation compared to DNA, which lacks this 2'-OH group, making it more stable and less reactive under similar conditions. ○ This difference is crucial for their biological functions: DNA's stability makes it well-suited for long-term storage of genetic information, while RNA's relative instability allows it to function effectively in transient roles, such as carrying messages (mRNA) or catalyzing reactions (ribozymes). DNA is composed of negatively charged phosphate group, a ribose sugar, and the nitrogenous base. The nucleotides are connected from one another from the oxygen of the 3’-end to the phosphate in the 5’-end of another nucleotide.  LESSON # 1: Central Dogma of Molecular Biology ○ Phosphodiester bond: Bond between each nucleotide. Why is having a phosphate backbone efficient? ○ MOTILITY: Phosphate groups are naturally negatively charged. This allows them to move freely in the cell as they are highly soluble in water. High solubility in water are essential for interactions with proteins, enzymes and other cellular components. ○ STABILITY: Like charges repel, unlike charges attract. The negatively charged phosphate group repels all the other phopshate groups from the other nucleotides which helps it keep its linear double helix structure which is useful for replication, transcription, and translation. ○ FAMILIARITY: Phosphate groups are already abundant in the cells which means that our cells already know how to efficiently handle them. (e.g. ATP, ADP, AMP) Why did our cells become efficient in handling phosphate groups?  LESSON # 1: Central Dogma of Molecular Biology ○ Prebiotic chemistry and chemical evolution (Sir’s topic of interest). When life first emerged on earth, the world is abundant with phosphate. As time went on, pre-historic cells has learned how to efficiently use this for their own gain. Once it achieved that, conservation through evolution in which evolution will always favor this chemically stable and efficient system. Unidirectionality of nucleotides  LESSON # 1: Central Dogma of Molecular Biology DNA has polarity. The direction on how to read it matters. Why is it read from left to right and not from right to left? Because it is the convention. DNA has polarity. It’s always wrote and read from the 5’ to 3’. The 5’ end always has a free phosphate group while the 3’ end always has a free hydroxyl group. Double-stranded DNA is anti-parallel. The other side goes from 5’ to 3’ from the other strand goes from 3’ to 5’. The bases are complementary. The bases on the 5’ to 3’ are complementary with the 3’ to 5’. DNA is helical in shape. It is a double helix as discovered by Watson and Crick.  LESSON # 1: Central Dogma of Molecular Biology Why should it be helical in shape? Helical structures reduces the electrostatic repulsion between the negatively charged phosphate backbones. If it is linear in structure, the sugar-phosphate backbone are aligned directly on top of one another and malapit ‘yung proximity nila sa isa’t-isa which increases the electrostatic repulsion. Base pairs are connected thru hydrogen bonds Adenine and Thymine creates double hydrogen bonds. Cytosine and Guanine creates triple hydrogen bonds. 40:01-1:20:00: Alfonso Q: Why is the percent concentration of G-C pairs important in primer design? ○ A: For thermal stability and its melting temperature. Q: How many percent should the G-C concentration be? ○ A: 40% - 60% GC content is the optimal range. Q: What will happen if the G-C content is only less than 40%?  LESSON # 1: Central Dogma of Molecular Biology ○ A: Pwedeng madegrade yung mga molecules natin kasi mataas po yung temperature ng PCR Q: What will happen if the G-C content is more than 60%? ○ A: Baka mahirapan maamplify sa PCR machine ○ A: Baka mag hairpin loop and ayun yung magcocause na mahirapan ○ A: Baka magkaroon ng non-specific binding ○ A: Kasi yung pagcalculate ng melting temperature is associated sa annealing step so kapag mali yung temperature, mali rin yung annealing step. Therefore, hindi nag bind yung DNA polymerase sa template ○ A: The primer is so stable na hindi siya madedenature kahit na mataas yung temperature. Kapag tinaasan yung temperature, madedenature lahat bonds. Lower concentration of GC (60%) will have a harder time to denature those bonds. Additional: The bond will become so sticky that it will not anneal. Q: Why do base pairs use hydrogen bonds? ○ A: Para enough lang stability for the DNA to hold its sheet, and hindi rin dapat ganun kalakas yung bonds para during DNA replication, maghiwalay yung dalawang bonds Additionals: During DNA replication, hydrogen bonds between base pairs are broken to separate the two strands of the DNA double helix. This allows each strand to serve as a template for the synthesis of a new complementary strand. Additionals: After replication, hydrogen bonds re-form between the complementary bases of the newly synthesized strands, ensuring that the double-stranded DNA is restored. Additionals: Other bonds are too stiff that it will be harder for them to be separated. ○ A: hydrogen bonds are bonds that can stabilize secondary structure.  LESSON # 1: Central Dogma of Molecular Biology Efficient Replication System ○ Single stranded → TCATT ○ Added stranded → AGTAA Semi-conservative replication ○ Half of the DNA came from the original template strand, and the other came from the new strand. ○ We conserve the original copy of the parental copy of the gene ○ Semi, half of the strand is conserved. Central Dogma of Molecular Biology DNA is just the repository for all the information and instruction on how to make proteins. Proteins are machinery inside the cell that actually affects and do things. Transcription ○ We have the double stranded DNA and then we make a single stranded copy of it, which is the mRNA. ○ The transcribed RNA copies are used to translate it to RNA?  LESSON # 1: Central Dogma of Molecular Biology ○ Q: Why do we still have to transcribe it to RNA for it to translate to proteins? A: Transcription happens outside the nucleus (cytoplasm), so DNA needs to be transported first. A: DNA needs to be in a single-stranded structure (mRNA), since a ribosome can only read single strands for it to translate into proteins. A: Because of the introns and exons. Hindi pwede na yung DNA matranslate directly into proteins masasama yung introns and exons, which are non-coding regions. A: It would expose the cell to cellular stress, causing mutations. ○ mRNAs act as the buffer genetic information between DNA and protein synthesis. That’s why evolution takes place that DNA must first be transcribed to RNA before it is translated to proteins. mRNAs deliver information from the DNA to build proteins. Hence, it’s name; It acts as the messenger. ○ So as we know the process of RNA is called transcription. ‘Scribes’ → people who copy documents before In transcription, we make an mRNA copy of the specific gene sequence that we want. (See Photo below) The purple binds to the promoter region of a specific DNA sequence The mRNA copy of the gene, which is complementary to the gene sequence, is created. Example: If the specific sequence that you want to read is “GTAAGC”, the mRNA copy is “CAUUCG”  LESSON # 1: Central Dogma of Molecular Biology Translation ○ The language used is amino acid. Amino acids are the building blocks of proteins ○ mRNAs are being translated into a fully functional protein. In the language of proteins, there are about 20 amino acids or “letters” ○ If only one pair of base pairs are used, it will only produce 4 amino acids. (41 = 4)  LESSON # 1: Central Dogma of Molecular Biology ○ If two base pairs are used, it will only produce 16 amino acids. (42 = 16) ○ If three base pairs are used, it will produce 64 amino acids. (43 = 64) Q: Why are there 64 amino acid codons if there are only 20 amino acids? ○ A: The reason why there are excess codons is because we need stop codons that could stop the translation. ○ A: Multiple codons prevent mutations para kapag nagkaroon ng mutation sa isang codon, meron pang maayos na codon. The 64 possible combinations of codons allow silent mutation. ○ Silent mutation - a mutation where in the principle of redundancy in the genetic code allows the mutation of nucleotide sequence to not change the resulting amino acid ○ For example: Let’s say that the codon code for “Lysine” is AAA, but mistakenly, the coded codon became AGA. Based on the Codon table, AGA is still a “Lysine.” ○ This is one of the evolutionary safeguard/advantages of the gene. 1:20:01 - END: Fenis  LESSON # 1: Central Dogma of Molecular Biology All proteins start with the amino acid methionine (AUG). thats why methionine is also called the start codon Meanwhile, there are three stop codons: UAA, UAG, UGA ○ Q: Bakit tatlo yung stop codons A: Naging part evolution. Dahil sa redundancy ng pagevolve ng mga codons at para maiwasan ang mutations kaya mayroong tatlong stop codons. ○ Q: Ano yung pine-prevent? Bakit tatlo yung stop codons A: Premature termination A: Premature termination. At ang pinaka-importante ay hindi materminate yung protein paulit ulit–that causes intense growth, which causes cancer. Once the ribosomes read the stop codons, it will stop the translation called translational termination. Translation is the process where the ribosomes will scan for the start codon (AUG) and whatever codon it will meet, it will translate into the corresponding amino acid until it reaches the stop codon DNA Replication Essential for cell replication ○ Growth, development, repair, regeneration, and maintenance ➔ If the cell replicates, then the DNA will be replicated as well S-phase or synthesis phase ○ Q: Sa cell replication may phases. G1, S phase, G2 phase and then mitosis which is divided into four phases. In which of these steps or phases does DNA replication take place? A: S phase or Synthesis phase Semi conservative replication ○ Q: Ano ulit meaning the semi-conservative? A: kalahati lang galing sa parent A: Naconserve parin yung sa parent. Semi means half lang yung nabago  LESSON # 1: Central Dogma of Molecular Biology A: Only one parent strand is needed to make a new one. Conserving part is the one strand of the parent to make a new one. Unidirectional replication (5’ to 3’) ○ It means that the DNA polymerase, it will add the nucleotides to the 3’ end of the parent strand ○ Q: Ano yung functional group na nasa 3’ end? A: Hydroxyl group of the sugar molecule ○ Q: If hydroxyl group yung nandoon, ano ang idadagdag ni DNA polymerase. What functional group? Phosphodiester bonds occur between the hydroxyl group and the? A: Phosphate group Initiation, Elongation, and Termination DNA polymerase ○ The major protein or enzyme involved in DNA replication is DNA polymerase ○ Q: From the name itself, what do you think is the function of a polymerase, hindi lang specifically DNA polymerase A: Nagsi-synthesize ng DNA strands. Siya yung nagda-dagdag ng polynucleotide. DNA Transcription To make an RNA copy of a specific gene sequence from DNA which will be used to make proteins Accurate measure of gene expression  LESSON # 1: Central Dogma of Molecular Biology ○ Q: Why RNA is an accurate measure of gene expression. Bakit name-measure ang gene expression sa levels of mRNA, not sa levels of DNA? A: Kapag gagawa ng protein, need ng mRNA. then , yung gene expression ng differentiated cell ay magkakaiba dahil magkakaiba ang functions nila. A: yung mRNA yung product mismo ng transcription and yung first step ng gene expression. Because of that narereflect yung activity ng gene tsaka kung gaano karami yung natatranscribe ng mRNA A: yung mga genes are present in multiple copies but may not necessarily actively transcribe A: Kasi sa cells natin lahat may specific genes. Mame-measure lang yung gene expression if yung specific cell ay mayroong mRNA copy for that specific gene. It means yun lang yung gusto iexpress na genes ng cell. Mayroong specific cells na ayaw naman iexpress yung ibang gene. So name-measure yung gene expression through the quantification of mRNA copies na nandoon. ○ Q: Yung DNA expression sa eukaryotes, san siya nangyayari? Saan yung DNA transcription sa prokaryotes A: Inside the nucleus for eukaryotes. Cytoplasm for prokaryotes It occurs in the nucleus and exit through the nuclear pores in eukaryotes, cytoplasm in prokaryotes Main functional proteins involved in transcription are RNA polymerase and spliceosome ○ Q: What do you think is the function of the spliceosome A: Related sa splicing. catalyst or removal of introns sa RNA. ○ Q: Kini-cleave lang ba ng spliceosome yung introns or may iba pa siyang ginagawa A: After removing introns, jino-join niya ulet yung exons which are the coding regions  LESSON # 1: Central Dogma of Molecular Biology A: Hindi lang kini-cleave, kase kapag nagcleave lang isya, mahahati sa gitna, mayroon ka lang dalawa. So jino-join din niya yung coding regions or exons. ○ Q: What is the difference between introns and intergenic regions? A: Introns ay within DNA sequences, intergenic regions are non-coding regions between DNA sequences Initiation, Elongation, and Termination Polyadenylation ○ Before it leaves the nuclear pores, after the mRNA transcript is made, polyadenylation occurs–the addition of a chain of adenine nucleotides in the 3’ end of the mRNA or the poly(A) tail that consists of 250 bases full of adenine. ○ Q: What is the relevance of the polyadenylation step? A: Nagse-seal and nagpe-prevent ng degradation once lumabas na yung mRNA ○ Q: How does polyadenylation prevent degradation? And degradation from what? If the mRNA is important, bakit siya nadedegrade ng cell? Clue: It is one of the differences between eukaryotic and prokaryotic DNA transcription A: the poly(A) tail ay nagco-connect sa 3’ end of the mRNA transcript. In the first place, ang mRNA is not supposed to be in the cytoplasm. So idedegrade siya ng exonucleases, enzymes outside of the nucleus. And yung mga RNA constructs ay nirerecycle for future use. So paano nagkakaroon ng distinction? Hindi basta RNA ay idedegrade agad. Si poly(A) binding proteins ay nasa labas ng nucleus, sa cytoplasm. Kapag lumabas si mRNA with poly(A) tail or poly(A) binding proteins (PABPs) 1-5 and na-meet niya si exonuclease, hindi siya ice-cleave since nandoon na si PABPs na sinasabing protection ng mRNA natin. ○ Q: Bakit din nandoon si exonuclease  LESSON # 1: Central Dogma of Molecular Biology A: Kasi may mga mRNAs na mali yung pagkakalabas, mali yung transcript, kaya nadoon si exonuclease. To counter that, nandoon din si PABPs. Protein Synthesis/ Translation Final step or ultimate step The central dogma of molecular biology only describes how information or instructions flow in order to make proteins ○ Q: Bakit ba kailangan ng proteins A: Because virtually, almost all cellular functions are facilitated by proteins or involved ang proteins. From structure to metabolism, to enzyme. ○ Q: Can you give me an example of protein na involved sa cellular structure? A: Actin. A: Actin or cytoskeleton, microfilaments, microtubules, etc. A: Addition molecules such as cadherins and integrins ○ Q: Proteins involved in metabolism? A: Enzymes na nagpapatakbo ng mga glycolysis or krebs cycle. ○ Q: Can you give me one enzyme involved sa glycolysis? A: Hexokinase that facilitates conversion of glucose to glucose-6 phosphate A: Others include pyruvate kinase and such ○ Q: Can anyone mention one enzyme involved in krebs cycle A: Acetyl coA carboxylase na nag cacatalyze ng conversion ng acetyl coA to malonyl coA A: Aconitase (For isomerization of citrate to isocitrate via cis-aconitate) ○ Q: Anong enzyme yung pinaka-unang step? Well its a cycle, so walang first step. Pero ano yung pinakaunang step sa citric acid cycle? A: Citrate synthase  LESSON # 1: Central Dogma of Molecular Biology ○ Q: Bakit siya tinawag na citric acid cycle? Bakit siya tinawag na tricarboxylic acid cycle. Bakit siya tinawag na cycle? A: Citric acid cycle: citric acid yung first compound na naform sa cycle Krebs cycle: pinangalanan siya kay hans krebs, nagintroduce ng pathway Tricarboxylic acid cycle: kase presence ng tricarboxylic acid yung intermediate (Note: Pero actually and in fact, tricarboxylic acid cycle siya dahil citric acid is a tricarboxylic acid. Synonymous sila.) Cycle: krebs cycle, paikot ○ Q: Pano narerecreate yung last step into first step ulit A: Formation of citrate, magiging oxaloacetate, magiging citrate siya ulit. Occurs in the cytoplasm. Specifically in the ribosome found in the cytoplasm (free-floating or RER) ○ Q: Bukod sa RER, ano pang part ng cell ang studded ng ribosomes. Clue: continuous yung RER sa part na ito. Kaya tinawag na rough kasi kapag tinignan sa microscope, yung texture ng RER is rough dahil studded siya ng ribosomes A: Nuclear envelope A: Outer layer or membrane of nuclear envelope ○ Q: Kung protein ang ginagawa ni RER, ano naman ang sine-synthesize ng SER? Clue: macromolecule A: Lipids rRNA and tRNA Initiation, Elongation, and Termination (fourth step which is ribosome recycling)