Molecular Biology Lecture Notes PDF

MOLECULAR BIOLOGY | Topic 2 | MLS 420 - LEC P2: REVIEW ON NUCLEIC ACIDS & PROTEINS Professor: Milliem Ruzzlee S. Reyes, MD Date: February 2, 2023 THE CENTRAL DOGMA NUCLEIC...

MOLECULAR BIOLOGY | Topic 2 | MLS 420 - LEC P2: REVIEW ON NUCLEIC ACIDS & PROTEINS Professor: Milliem Ruzzlee S. Reyes, MD Date: February 2, 2023 THE CENTRAL DOGMA NUCLEIC ACIDS NUCLEOSIDE NUCLEOTIDE The central dogma involves the transfer of genetic information from DNA to RNA through transcription, and then Nitrogenous base + NucleoSIDE + PhosphaTe from RNA to protein through translation. This flow of Pentose SUGAR information is fundamental to the synthesis of proteins, which It is held together by: It is held together by: are essential for the structure and function of living N-glycoSidic bond EsTer bond organisms. Nitrogenous bases - these are your Purines and Pyrimidines SUMMARY OF THE PROCESS: Your phosphate groups are held together by ANHYDRIDE BONDS DNA (Deoxyribonucleic Acid): The genetic material that carries hereditary information in cells. PuGA = Purines, Guanine & Adenosine (in DNA and RNA) ↓ CUTiePy = Pyrimidines, Cytosine, Uracil (RNA), & Thymine Transcription: The process in which (DNA) information from a DNA sequence is transcribed into RNA. This occurs in the cell nucleus. ↓ RNA (Ribonucleic Acid): The single-stranded nucleic acid molecule that serves as a messenger carrying genetic information from DNA to the site of protein synthesis. ↓ RIBOSE DEOXYRIBOSE Translation: The process by which the information in RNA is used to D-ribose sugar in RNA 2’-deoxy D-Ribose sugar in build a protein. It takes place in the DNA ribosomes of the cell. RNA has its HYDROXYL GROUP (OH) ↓ DNA has no hydroxyl group (H only) Protein: The end product of the central dogma, synthesized through translation. Proteins play crucial roles FUNCTION OF NUCLEOTIDES in the structure and function of cells, tissues, and Building blocks of nucleic acids (DNA/RNA) organisms. Other functions: ○ Carriers of activated intermediates ○ Structural component of coenzymes Like in your lipid pathways or your pyruvate (kreb’s cycle) ○ Second messengers in signal transduction pathways When you have a receptor in your cell membrane which receives stimulus and the second messengers (cAMP) inside the @mlstranses | 1  cell passes the message received HISTORY unto the nucleus of the cell. ○ Principal biological transducers of free energy E.g. ATP ○ Regulatory compounds in pathways Rate limiting step in glycolysis (Phosphofructokinase 1) ○ Synthetic analogues as drugs Like drugs given to pregnant women who have HIV such as: Zidovudine Another example are drugs given to patients with covid 19 such as: Remdesivir 1. Composition of DNA was known 2. Erwin Chargaff’s experimental finding: Chargaff’s A. DEOXYRIBONUCLEIC ACIDS (DNA) rule a. DNA base pairing b. Your purines will always be in equal number with your pyrimidines c. A-T pair has 2 hydrogen bonds (weaker); G-C pair has 3 hydrogen bonds (stronger) 3. Rosalind Franklin obtained X-ray crystallography images of DNA 4. James Watson and Francis Crick created the two-strand, double-helix model STRUCTURE AND PROPERTIES Made up of nucleotides held together by 3’ to 5’ phosphodiester bonds Consists of 2 chains ○ Complementary (Chargaff’s rule) ○ Antiparallel (3‘ - 5’ the other 5’-3’) ○ Hydrogen bonded by the bases In 1920, there was a group of scientists (they still didn’t know Right-handed double helix (usually) what the structure of DNA looked like). They were trying to 10.5 base pairs per turn (usually) figure out what thing in the body contained the info that we pass on to the next generations. They took S strain and R strain of a certain bacteria and then they got a substance from the S strain then they put it in the R strain (They didn’t know what that something was, but it was from the S strain). As time went by, they realized that their cultures of the R strain, transformed to the S strain and they survived in the culture. So they concluded from that experiment that there must be something from the substance from the S strain which holds the genetic information of the strain. In 1940s, they purified the substance S strain substance into different macromolecules (RNA, protein, DNA, Lipid, Carbohydrate) and then they put it in the R strains again, and then they saw, that the only ones who transformed into the S strain was those who were given the DNA. That is where they realized that DNA holds the secret to life. People were now interested in studying DNA. Bases of the DNA are projected inward in-order for them to be safe and to be bonded together @mlstranses | 2  A-DNA* B-DNA Z-DNA** # of BP per 11 10 12 turn Morphology Broad & Long & Long & Thin Short Thin Screw Right Right Left-handed sense handed handed Features In low Most humidity & In 5’ end of common high salt chromosomes form conditions *A-DNA condenses because of high salt conditions where there is less water; **found in areas where there are consecutive GCGCGC LEVELS OF DNA ORGANIZATION 1 Minor grooves - backbones are closer together 2 nm DNA Double Helix Major grooves - backbones are far from each other Will eventually loop around a histone octamer Grooves are important to note because it is where your DNA and protein interactions happen BASE STACKING 2 10 nm chromatin fibril Histone + DNA wrapped around it = NUCLEOSOME Made of nucleosomes separated by LINKER DNA DNA wrapped 1.75x over a histone octamer (left-handed) 3 The bases resembles a staircase; it is because of these 2 interactions: ○ Hydrophobic interactions 30 nm chromatin fibril (Solenoid) The bases will cluster inside since When many nucleosome group together in a 30 nm they are hydrophobic in nature chromatin fibril it is now called a Solenoid ○ van der Waals forces These forces help maintain the 4 Supercoiled structure structural integrity of the DNA double helix by stabilizing the Promotes packing DNA into compact structures close packing of the bases along To save space it should be tightly packed the helical axis. The distance between one atom 5 Chromosome and another atom so that they Condensation of DNA during PROPHASE of mitosis don't overlap 23 pairs of chromosomes (46 total) ○ 22 pairs autosomes ○ 1 pair of sex chromosomes @mlstranses | 3  ○ The cap serves as a guide to the ribosome ○ The tail serves as a protection Primary transcript undergoes splicing prior to protein synthesis ○ Exons are retained (expressed) NON PROTEIN-CODING A. Ribosomal RNA (rRNA) Most abundant RNA (80% of total RNA) Ang rami-raming rRNA! HeteroChromatin = Highly Condensed Euchromatin = Expressed Contributes to the formation and function of ribosomes HETEROCHROMATIN Contain many loops and BASE PAIRING Condensed, darker on EM Molecules differ in their sedimentation Sterically inaccessible coefficients: Transcriptionally INACTIVE ➔ Prokaryotes: 50S and 30S subunits, ↑ METHYLATION; ↓ ACETYLATION made up of 3 types of RNA: 16S, 2S and 5S ➔ Eukaryotes: 60S and 40S subunits, EUCHROMATIN made up of 4 types of cytosolic Less condensed, lighter in EM rRNA: 18S, 28S, 5S and 5.8S Sterically accessible B. Transfer RNA (tRNA) Transcriptionally ACTIVE Smallest RNA (15% of total RNA) ↓ METHYLATION; ↑ACETYLATION Tiny tRNA Adapter molecules that translate Methylation and Acetylation are epigenetic modifications nucleotide sequences of mRNA into meaning they dont happen on the DNA itself; they happen specific amino acids on your histone 74 to 95 nucleotides with high % of - In Methylation, if you have more methyl groups it unusual bases Mutes your DNA; nagiging inactive At least 20 different species - In Acetylation, if you have more acetyl groups it ○ Because there are 20 amino acids ○ 1 tRNA for 1 amino acid Activates your DNA; nagiging active Contains an anticodon ○ Connects the codon to a B.RIBONUCLEIC ACIDS (RNA) corresponding amino acid or a stop codon (UAG, UAA, UGA) Similar to DNA with purine and CLOVERLEAF appearance in 2D pyrimidine bases (they have a Uracil (Acceptor arm terminates the nucleotides –CCA and instead of a Thymine) receives the tRNA-appropriate amino acid) Single stranded C. Small Nuclear RNA (snRNA) FUNCTIONS depend on the type Functions in mRNA processing and rRNA processing and can be: Splice together the EXONS to form the ○ Coding mature mRNA ○ Non-coding D. Micro-RNA (miRNA) Acts as an interference PROTEIN-CODING Interact with the 3’ untranslated region of A. Messenger RNA (mRNA) mRNA to induce mRNA DEGRADATION and TRANSLATIONAL Most heterogeneous RNA (5% of total RNA) REPRESSION mRNAs are magkakaiba ○ They can also sometimes interact with other parts of your mRNA Conveys information from DNA to the (uncommon) translation machinery (ribosomes) ○ Others can also activate translation Template for PROTEIN SYNTHESIS yet it is very uncommon In eukaryotes, it has a methylguanosine cap at the 5’ end and a poly(A) tail at the 3’ end @mlstranses | 4  E. Long Non-Coding RNA (lncRNA) Double-stranded RNA (20-24 bp) Interfere with the expression of genes that have Non-coding transcripts of >200 nt complementary nucleotide sequence to that of Involved in regulation of cell differentiation & development, and siRNA maintenance of telomere length (TERC ○ It implies that we can use siRNA to target and TERRA) specific genes; which can be used for ○ When the chromosome replicates therapy because it is very specific over and over again, the telomere Induces mRNA degradation gets shortened; that shortening of the telomere is one of the theorized reasons on why we age DNA RNA and why we eventually die. ○ That is also the thing that allows Sugar your cancer cells to remain alive Deoxyribose Ribose moiety (they have their own telomerase which elongates their own Purines Adenine & Guanine telomeres) TERC - Telomerase RNA Component Pyrimidines Cytosine & - Acts as a template for the synthesis Cytosine & Uracil Thymine of your telomeres TERRA - Telomeric Repeat-containing RNA Structure Double-stranded Single-stranded - RNA component of your telomeres F. Silencing RNA (siRNA) Chargaff’s Applies Does NOT apply rule Stability Stable Unstable Not hydrolyzed Can be by alkali d/t hydrolyzed by absence of 2’ alkali to hydroxyl group 2’,3’-cyclic diesters of the mono- nucleotides DNA is more stable because it lacks the hydroxyl group; withstands the hydroxylation by alkalis ○ In order for you to be hydrolyzed you need a hydroxyl group and since the DNA has no hydroxyl groups it cannot be hydrolyzed. C. PROTEINS THE PROCESS OF siRNA - Your double stranded RNA (dsRNA) or your hairpin loop they get spliced by your DICER PROTEIN, that now creates your siRNA DUPLEX. Then that siRNA with the ARGONAUTE PROTEIN (a family of protein which has a splicing capability) complexes to form RNA Inducing Silencing Complex (RISC), then this RISC will now start looking for the Most abundant and functionally diverse complementary strand to your siRNA molecules in living systems Linear polymers of amino acids When that RISC finds its complementary strand, it ○ N-CX-COOH (X: variable R group or attaches forming a siRNA-mRNA complex and since the sidechains) mRNA is bound to a siRNA the mRNA is now silenced ○ Linked together by PEPTIDE BONDS and it cannot be translated. Then because there is something that is attached in your mRNA, your body recognizes it as foreign so the mRNA gets degraded in the long run. @mlstranses | 5  SIDE CHAINS Lysine Arginine Histidine Cysteine can form disulfide bonds because they contain sulfur ○ Methionine has sulfur but it is not as accessible as the sulfur in the cysteine because it is in the middle Glycine & Proline can affect the protein structure Glutamate ○ Glycine has a side chain that is so small Aspartate which disrupts the protein structure; proline on the other hand has a big side chain which can also disrupt the protein structure E.g. OXYTOCIN (9 AAs; Smallest AA) Between 100-1000 AAs in length ○ Longest protein is TITIN (25,000 AA) Found in our muscles that stabilizes our thick filaments Protein sequence can be determined by removing one AA at a time (Edman degradation) Asparagine PROTEIN FUNCTIONS Glutamine ➔ Regulate metabolism Serine ➔ Facilitate muscle contraction Threonine ➔ Provide structural framework Tyrosine ➔ Shuttles molecules in the bloodstream ◆ Usual shuttle in the body is the ALBUMIN ◆ Hemoglobin is also a shuttle ➔ Components of the immune system @mlstranses | 6  STRUCTURAL ORGANIZATION OF PROTEINS 2 SECONDARY STRUCTURE The folding of short (3-30 residues) segments of polypeptide into geometrically ordered units Stabilized by HYDROGEN BONDS MOTIFS - supersecondary structures produced by packing of side chains from adjacent secondary structural elements ○ Example is the PORINS of a bacteria, they sometimes use it as an antibiotic resistance mechanism which serves as channels or pores through which molecules can pass. 1 PRIMARY STRUCTURE Determined by the AA sequence Has an N (NH3) and a C (COOH) terminus ALPHA HELIX ○ N = eNtry (targeting signals) Most common secondary structure ○ C = Contain (retention signals) Spiral with polypeptide backbone core and side chains extending OUTWARD Peptide bonds attach the α–amino group of one to the ~3.6 AAs per turn α–carbonyl group of another An example of a 100% α-helix is our KERATIN & ➔ Partial double-bond character HEMOGLOBIN ➔ TRANS - configuration ➔ Can be disrupted by hydrolysis ◆ Because your peptide bonds are created by dehydration reax; you are taking out water from to form your peptide bonds ◆ Therefore if you add water (strong acid or base) into it, it will cause hydrolysis; it will break apart BETA SHEET AA residues form zigzags or a pleated pattern R groups of adjacent residues project in OPPOSITE directions Can be parallel or antiparallel @mlstranses | 7  An important Beta-Sheet that is nice to remember is in the Alzheimer’s disease; in the senile plaques, the neurofibrillary tangles accumulate in the brain which causes degeneration Immunoglobulins are also made up of Beta-Sheets 4 QUATERNARY STRUCTURE 2 or more polypeptide chains forming one 3 macromolecule Not all proteins have a corresponding quaternary structure CLINICAL CORRELATE: SICKLE CELL DISEASE TERTIARY STRUCTURE Overall 3D shape of the protein Stabilized by: ○ Hydrophobic clustering force ○ Disulfide bridges (Cysteine) ○ Hydrogen bonds (between your polar side chains) ○ Ionic interactions (between your charged side chains) ○ van der Waals forces (to avoid overlapping) Due to a point mutation (missense) in both genes coding for the β-chain Change from a Glu → Val at position 6 ○ This change is very significant because Glutamate is an ACID and Valine is a NON-POLAR side chain which presents a PROTEIN FOLDING big change in the AAs and also the final The AA sequence holds all the info needed conformation of your protein because non Proteins fold into a conformation of LOWEST polar side chains don't have a charge ENERGY ○ The final folded form of your proteins is HOMOZYGOUS RECESSIVE disorder the form that the body made using the lowest energy requirement EFFECTS ON RBCs ○ That is the reason why usually our proteins has only one stable conformation Most proteins fold to a single stable conformation MOLECULAR CHAPERONES assist in protein folding but are NOT the determinants of the final structure; Hasten the process of the folding before they get degraded They take those partially folded proteins and hasten the process; they cannot just take proteins that are completely unfolded and then fold them. Example of a molecular chaperone is Hsp70 PROCESS: helps misfolded proteins fold into the right Polymerization and decreased solubility of the deoxy form of conformation. Hb in LOW OXYGEN tension (Low affinity for oxygen) → Distortion of RBC membrane → Sickling of RBCs (Very Rigid and not flexible) → Occlusion of capillaries @mlstranses | 8  CLINICAL MANIFESTATIONS Anemia ○ Low affinity for oxygen; Abundant HbS than HbA Tissue anoxia ○ Less to no oxygen delivered in the tissues Painful crises ○ Because of those occluded vessels Treatments Hydration Analgesics ○ for the pain Antibiotics* ○ If they have infection Transfusions Hydroxyurea ○ Counteracts the low affinity of your HbS to O2 by increasing the production of HbF (Fetal Hemoglobin) which has higher affinity to oxygen L-glutamine ○ Reduce oxidative stress ○ Reduce the damage Crizanlizumab ○ Monoclonal antibodies ○ Inhibits p-selectin Helps reduce the chances of vessel occlusion Voxcelator ○ Changes affinity of HbS to O2 to increase its affinity @mlstranses | 9