Replication, Transcription, and Translation PDF

REPLICATION, TRANSCRIPTION, AND TRANSLATION Strands LECTURER: Ms. Rianne Aubrey P. Lopez, RMT ○ DNA: Double stranded ○ RNA: Single st...

REPLICATION, TRANSCRIPTION, AND TRANSLATION Strands LECTURER: Ms. Rianne Aubrey P. Lopez, RMT ○ DNA: Double stranded ○ RNA: Single stranded COURSE OUTLINE A. DNA AS A TEMPLATE I. Central Dogma of Molecular Biology A. DNA as a Template For a molecule to serve as a genetic material, it must exhibit B. Central Dogma II. DNA Replication the following crucial characteristics: A. Models B. Enzymes Capable of replication C. Replication Process ○ No matter what, DNA must replicate because D. Eukaryotic Replication some of it will die out and it needs to create III. DNA Transcription more. It also needs to adapt and change for A. Transcription Process an individual to survive. B. Post-Termination and RNA Processing Storage of information IV. Kinds of RNA A. Major Kinds of RNA ○ There are many types of storage: B. Minor Kinds of rna ○ Example: Melanin. Sometimes melanin V. DNA Translation activates, sometimes it doesn’t. Sometimes A. The Genetic Code you have melanin that will be shown, B. Translation Process sometimes it doesn’t. C. Post Translation ○ It stores that information until it is going to be VI. Genetic Mutations used. Repository in a sense. A. Molecular Types Expression of information B. Effects when Translated into Proteins ○ Phenotypic characteristics or observable characteristics. LEARNING OBJECTIVES Variation by mutation Understand the significance of Central Dogma Determine roles of different enzymes in DNA Replication This serves 2 purposes: Discuss the stages of DNA Replication Discuss steps in DNA Transcription Source of information for protein synthesis Differentiate Introns from Exons ○ Which is transcription. Explain the process of RNA processing, modification, and splicing Provides information inherited by daughter cells Enumerate and understand the different types of RNA Discuss the process of translation Note: Until 1944 it wasn't clear what is the component in our Correlate relationship of code defects to corresponding mutations body, that is our genetic material, until different experimentations occurred. Lots of these experiments came from the I. CENTRAL DOGMA OF MOLECULAR BIOLOGY experimentation of a specific bacteria. DNA B. CENTRAL DOGMA Also known as Deoxyribonucleic Acid, is the raw Describes the flow of genetic information in all of life. material of inheritance. This is a template as to what Dogma = truth makes you, you. Without it, we would be nothing. And with too much DNA, we would be something extra. These are bound mostly by 5’ to 3’ phosphodiester bonds. Composition: ○ Phosphoric Acid ○ Sugar: 2-deoxy-D-ribose ○ Nitrogenous bases Purines (Adenine and Guanine) Pyrimidines (Thymine and Cytosine) DNA vs RNA Name itself Sugar ○ DNA: 2-deoxy-D-ribose ○ RNA: D-ribose 1 Process of Central Dogma Semi-Conservative model 1. Everything starts with DNA. DNA is located inside the This was discovered by Matthew Meselson and nucleus. For DNA to become an amino acid, it has to Franklin Stahl in 1958 through experimentation with E. undergo different processes. coli (Escherichia coli) DNA 2. The DNA replicates itself to have multiple copies. Each daughter strand is only half of a new DNA (has 3. The DNA undergoes transcription. Through this, it can both old and new DNA) become multiple types of DNA. Most importantly, your One original strand is used as a template. mRNA. But it can also turn into your rRNA and tRNA. This is where our DNA lies because our DNA is not 4. As this becomes mRNA, it will undergo a process of always the same. It will change, it will continue on, RNA processing. that’s how we adapt in the world. It has new and old. 5. After which it will leave your nucleus, proceed to your ○ Old: because we need our memories and we cytoplasm and undergo translation into proteins and need our information from the past. amino acids. ○ New: because we need to adapt to the environment that we have today. How many chromosomes do we have in our body? SEMI-CONSERVATIVE MODEL DISPERSIVE MODEL 46 Chromosomes 23 Pairs Consistent strand of “new” Each strand is a mix of “new” and “old” and “old” II. DNA REPLICATION One strand is old A strand is new-old-new-old One strand is new The synthesis of a new molecule of DNA by copying existing DNA. B. ENZYMES INVOLVED ○ Needed in order to ensure that each new cell receives the correct number of Helicase chromosomes. This is how DNA copies itself into more than one Catalyzes the unwinding of double helix ahead of DNA strand because we can't just have one strand of DNA. polymerase We have multiples and these multiples have copies of It is the first enzyme involved because it will unwind each other to make sure we continue to live. the double helix. DNA starts as double, but they are replicated A. MODELS separately because the two strands will have different ways of replicating, depending on which direction they will be. Single Stranded Binding proteins or Helix Destabilizing proteins Also sometimes acronymed SSB Bind specifically to single stranded DNA to prevent reannealing. This one hates realignment. Hates pairs. This prevents any recombination because if DNA recombines before it is replicated, it will not replicate at all. Primase Conservative Semi-conservative Dispersive Model Model Model From the key word “Prime”, it catalyzes the synthesis The parental The parental of the short segment RNA primers (roughly 6-12 The parental strands stay strands are nucleotides long) to the template strands. strands do not together or dispersed into two It starts the process. reassociate reassociate new double helices DNA Gyrase or Topoisomerase Each strand of both Each replicated Each replicated daughter molecules DNA molecule is DNA molecule Relieves coiling tension ahead of the replication fork contains a mixture “conserved” as the consists of an ”old” and prevents supercoiling. of the old and new original and “new” strand As DNA tries to unwind, sometimes it will tighten, and synthesized DNA. the top portion of the DNA will be coiled, and will be Table 1. Models of DNA Replication tangled. This enzyme makes sure nothing tangles. Because if it The human DNA follows the semi-conservative model. tangles, replication stops prematurely, and you will have the wrong DNA. 2 DNA Polymerase III Involves four components (seen through electron microscope): Very major enzyme. It is the most important out of the ○ DNA helicase: separates double helix. three DNA polymerases. ○ Primase: initiates synthesis of an RNA It binds nucleotides to form new strands. molecule. Extends the strand in the 5’ to 3’ direction. ○ DNA polymerase: initiates nascent, daughter strand synthesis. DNA Polymerase I (Exonuclease) ○ SSBs: binds to single stranded DNA to make sure they do not realign and to prevent any Removes RNA primer and replaces it with DNA/inserts base pairing to happen. the correct bases. The replication fork is bidirectional: ○ One strand goes from the 5’ to 3’ (leading Ligase strand) ○ The other goes from the 3’ to 5’ (lagging Joins DNA fragments (Okazaki fragments) into strand) continuous daughter strands and seals other nicks in the phosphate backbone. C. REPLICATION PROCESS 1. Unwinding of DNA Strands Origins of Replication (Ori): a special site where replication begins. Single-strand binding proteins: will stick to each strand of DNA to make sure they will not come back together, and they will stay single through the process. From there, these are separated with the use of your helicase. ○ Helicase: makes sure that the strands separate. Imagine a zipper; when you pull it down, it separates into two separate strands of the zipper. With the formation of the replication fork, leading and lagging Then, your helicase proteins will bind to your DNA strands were mentioned: sequences, known as origins, which allow for processive unwinding of DNA Leading strand (Forward strand) The two strands here are now going to be your ○ DNA is synthesized continuously templates. These are your templates as to how this Lagging strand (Retrograde strand) will be replicated and the new strand will be elongated ○ DNA is synthesized into small fragments or by the hydrogen bonding throughout the process. Okazaki fragments (discontinuously). ○ It is called lagging strand because it lags behind the leading strand. Okazaki fragments Small fragments produced from the lagging strand. Produced discontinuously from a 5’ to 3’ direction Discovered in 1960 by Reiji and Tsuneko Okazaki whilst studying DNA replication through experimentation with E. coli Crucial in discovering what is the importance of DNA for humans. 3. DNA Polymerase Complex Polymerases: 2. Formation of Replication Fork The first purified enzyme shown to catalase DNA The Ori is where replication begins. Meanwhile, the replication was designated as DNA Polymerase (pol l) replication fork is where it is currently happening, DNA Polymerase II (pol Il) and Polymerase III (pol IIl) meaning it will continuously move. were later discovered. Replication fork: the bidirectional thing that represents Pol III has the most function compared to the pol I and where the replication is happening currently. pol II. Pol Ill is the main polymerizing enzyme during bacterial replication. Several different DNA polymerase molecules engage in DNA replication. 3 Why are these important? III. DNA TRANSCRIPTION Chain Elongation Copying of one strand of DNA into RNA by a process ○ accounts for the rate at which polymerization like replication. occurs. The synthesis of RNA under the direction of DNA, Processivity particularly mRNA. ○ an expression of the number of nucleotides added to the nascent chain before the Why do we need RNA? Why can’t we just copy DNA polymerase disengages from the template. completely? Proofreading function ○ identifies copying errors and corrects them, DNA only stores information. and an important function of DNA that is not It has to be transcribed into RNA to make amino acids in RNA. and proteins because DNA can’t make amino acids on its own. But, DNA can make sure we make the correct 4. Initiation and Elongation of DNA Synthesis RNA, so that the correct RNA can make the correct protein. Initiation requires a short length of RNA (roughly DNA does make proteins but it just needs to be RNA 10-200 nucleotides long) first. The replication complex begins to remove RNA primers. SIMILARITIES DIFFERENCES These gaps from the Okazaki fragments are now filled TO DNA SYNTHESIS TO DNA SYNTHESIS with the appropriate base-paired deoxynucleotide. The newly synthesized fragments of DNA are then Steps of initiation, Use of Ribonucleotides sealed through the action of DNA ligase. elongation, termination. Primer is not involved Large, multicomponent Uracil replaces Thymine as 5. Formation of Replication Bubble initiation complexes: base pair of Adenine ○ DNA: Uses DNA Only some portions of the As replication occurs, both strands are simultaneously polymerase complex genome are copied once replicated ○ RNA: Uses RNA This replication process generates Replication Bubbles polymerase complex Any nicks are resealed by DNA ligase. Adherence to Watson-Crick base pairing rules 6. Reconstitution of new chromatin structure ○ Adenine to Thymine (RNA: replace Thymine Newly replicated DNA is rapidly assembled into to Uracil) nucleosomes, the pre-existing and newly assembled ○ Guanine to Cytosine histone octamers are randomly distributed to each arm of the replication fork. A. TRANSCRIPTION PROCESS These are facilitated through the actions of Histones. RNA Polymerase Main enzyme involved in RNA transcription. Links ribonucleotides together in a sequence complementary to the DNA template strand. Catalyzes RNA synthesis. D. EUKARYOTIC REPLICATION Eukaryotic Replication Eukaryotic DNA replication is more complex than the replication process of a prokaryote (E. coli), but it is similar. Eukaryotes have large genome size, they have much more chromosomes than prokaryotes. In addition, the eukaryotic DNA is associated with histones and non-histone chromosomal proteins. Hence, eukaryotic DNA is replicated not as bare DNA but as chromatin. 1. Initiation 4  RNA polymerase and supporting accessory protein will bind to around 6-10 base pairs sequence of nucleotides on the DNA to be copied. The initial step in bacterial gene transcription is referred to as template binding. This region is known as the Promoter Regions or Initiation Sites. Once RNA polymerase has recognized and bound to the promoter, DNA is locally converted from its double stranded form to an open structure, exposing the template strand. ○ It will become single-stranded, but not RHO-DEPENDENT INTRINSIC TERMINATION completely. Only a small area will be opened, TERMINATION as opposed to replication where it completely Dependent on Dependent on the termination splits. self-complementary GC-rich factor, rho (r) and a sequences (inverted repeats) termination sequence that is within the transcript, which transcribed into a hairpin form a stable GC-rich hairpin structure in the transcript. structure, immediately followed by a string of uracil residues. Why is this important? This will vary its sequence and its ability to initiate transcription. From there, it will begin in the promoter region in a 5’ to 3’ direction. B. POST-TERMINATION and RNA PROCESSING The primary transcript, known as pre-mRNA, 2. Elongation undergoes extensive processing in the nucleus before being exported into the cytoplasm. As RNA polymerase moves down the strand and adds Contained within pre-mRNA, are sequences found in ribonucleotide complementary to the base sequence the DNA and mRNA known as EXONS and found in the of the DNA template strand (elongating the strand as DNA and not in the mRNA known as INTRONS. RNA transcript). The Sense strand of the template has a sequence identical to that of the RNA complex. The 5’ end of the synthesized RNA protrudes from the transcription complex 3. Termination Occurs once RNA polymerase reaches a specific termination sequence in the DNA. Sequences called terminators signal that the RNA transcript is complete. Once they are transcribed, they cause the transcript to be released from the RNA polymerase. 5  Addition of a methylated guanine nucleotide A. MAJOR KINDS OF RNA CAPPING (7-methylguanosine OR m7G cap) to 5’ end of RNA molecule. Process where introns are removed from the primary transcript and exons are bound together. SPLICING Splicing occurs within spliceosomes. Once completed, the mRNA travels from nucleus to cytoplasm. Coupled with mRNA rRNA tRNA transcription. messenger RNA ribosomal RNA transfer RNA Enzyme poly A polymerase adds 100-200 residues of adenylic acid to the end of 3’ end Contains and Forms the Transfer activated of the RNA transcript. carries the genetic ribosomes. amino acids to the TAILING code to the ribosomes to be Aid in export of mature mRNA. Render stability. cytoplasm for Largest used in assembling Serve as a recognition signal. controlling the type components of the protein of protein formed. RNA (80-90%) molecule. B. MINOR KINDS OF RNA snRNA / siRNA miRNA pre-mRNA Small Nuclear RNA Precursor Micro RNA / Splicing RNA messenger RNA Directs the splicing Single-stranded Has not yet of pre-mRNA to RNA molecules of undergone RNA form mRNA. 21-23 nucleotides processing. that regulate gene Helps in removing transcription and Its main difference introns. translation. from mature mRNA is that pre-mRNA Very small; support has not undergone IV. KINDS OF RNA the process. RNA processing. RNA TYPES ABUNDANCE STABILITY V. DNA TRANSLATION PROTEIN CODING 5 >10 different Unstable to mRNA 2% - 5% species very stable NON-PROTEIN CODING Large ncRNA 28s, 18s, 5.8s, 5s rRNA (found in 80% - 90% Very stable eukaryotes) Unstable to IncRNA = 1000s 1% - 2% very stable Small ncRNA RNA-directed synthesis of a polypeptide which involves: 60 different tRNA 15% Very stable mRNA species Ribosomal RNA (rRNA) 30 different Transfer RNA (tRNA) snRNA < or = 1% Very stable species Genetic Coding (Codons) mi/SiRNA 100s-1000 < or = 1% Stable Importance of DNA Translation mRNA is not yet a protein so we cannot use it, it has no purpose to us, and it is just holding the message so we need to translate that message into the protein that we need in our body. 6  A. THE GENETIC CODE B. TRANSLATION PROCESS Properties of the Genetic Code (explained further in lecture 2): Degenerate: multiple codons decode the same amino acid. Unambiguous: a single codon can only code for a single amino acid. Non-overlapping: no codons overlap in the reading. Without punctuation: the code is read continuously without punctuation. Universal: Applies to multiple (most) species 1. Initiation Starts with the interaction of mRNA, tRNA carrying the first amino acid of the polypeptide, and two subunits of a ribosome. A small ribosomal subunit binds to a molecule of mRNA. This signals the beginning of polypeptide chain Genetic Coding Dictionary sequencing. Four important parts of your translation: mRNA, tRNA, First suggested by Francis Crick rRNA, genetic Codons It has a pattern of degeneracy: Anticodon: are the 3 RNA bases that match the 3 ○ Example: UUA, UUG, CUU, CUC, CUA, CUG can bases of the mRNA molecule only code for Leu (Leucine) It starts in the interaction of your mRNA and tRNA, Francis Crick discovered the Wobble Hypothesis carrying the first amino acids ○ Suggest that the initial two nucleotides of the First it will search for your START codon (AUG), from triple holds more significance than the 3rd in there it will start translation. attracting the correct tRNA: Example: UUG : the first two letters, U and U are more important than the third letter G. In genetic mutations, silent mutation usually affects the third codon. Codons ALWAYS triplets of RNA bases. Determine which amino acid will be produced. There are 64 possible codons START codon (initiator) : AUG (Methionine) STOP codon : UAA, UAG, UGA 2. Elongation Once it catches the start codon, it will get the elongation factors. A large ribosomal subunit is where reading happens. As it moves along the line, it connects and as it moves away, ones that are not inside the ribosomal unit will be kicked out and those are now the new proteins. That’s how amino acids are formed. 7 Upon recruitment of an elongation factor and the large POSITIVE EFFECTS NEGATIVE EFFECTS ribosomal subunit, polypeptide chain synthesis progresses: Source of new alleles Cell death Codon recognition: anticodon of an incoming Genetic variation Genetic diseases (ex. aminoacyl tRNA base pairs with the complementary Selective advantage that cancer) mRNA codon. one specie gains over Peptide bond formation another Translocation A. MOLECULAR TYPES Substitution Mutation Occurs when a base at a certain position is replaced by another. From this, the substituted base will be paired with its appropriate base-paired partner. Transition ○ Pyrimidine to Pyrimidine or Purine to Purine Transversion ○ Pyrimidine to Purine and vice versa 3. Termination Occurs when a STOP codon in mRNA is encountered. Deletion Mutation ○ The stop codons are UAA, UAG, and UGA. It only needs to catch one stop codon to stop Occurs when one or more nucleotide pairs in a DNA and not all 3. molecule are lost or removed Upon recognition of the Stop codon, a release factor is given and interacts with the ribosome and polypeptide Insertion Mutations chain. It attaches to the Stop codon and basically tells the line that they’re done. Addition of one or more nucleotide pairs to the DNA ○ It will be clipped away and a new line of strand proteins is produced. From this, the large ribosomal subunits and release factor are removed and recycled to use with other Inversion Mutations mRNAs. ○ Elongation, From the start codon A complex 180-degree rotation of a segment of the (AUG/Methionine), large ribosomal units DNA without a loss or gain in nucleotide number recognize everything, moves along the line, and forms peptide bonds then once it finds a B. EFFECT WHEN TRANSLATED TO PROTEINS stop codon, it connects and then clips it out and recycles again. Silent Mutations (Synonymous) C. POST TRANSLATION No detectable change because of the degeneracy of the code. Most polypeptides undergo modification before they Mostly a change in the 3rd nucleotide of a codon. consider their final forms and functions. These forms (primary, secondary, tertiary, and quaternary) could be chemical change, folding, or formation of multi-subunit structures. Once folded, some are physiologically active as single subunits while others are then bound to other subunits to form Quaternary structures (e.g., Hemoglobin). VI. GENETIC MUTATIONS Mutations are heritable changes in the nucleotide sequence of DNA. Common misconception: mutations are bad. Mutations can be caused by many things: ○ Genetically: cause is natural, the body just makes mutations. ○ Environmentally: an example is radiation poisoning. In Chernobyl, most animals and plants in the area were exposed to high amounts of radiation to the point that they have mutations. 8 Missense Mutations (Non-Synonymous) Frameshift Mutation Occurs when a different amino acid is incorporated A frameshift is a shift in reading frame by insertion or into site of the protein molecule deletion of a nucleotide (may be one of the two or both) Acceptable Deletion and insertion of a single nucleotide alters the ○ Resulting protein molecule may not be reading frame. distinguishable. ○ Reading frames are done in codon triplets. ○ AAA → AAU Partially Acceptable ○ Will result in a protein molecule with partial but abnormal function. ○ GAA → GUA ○ The first nucleotide is considered to be the most important. However, due to the Wobble Hypothesis, both the first and second nucleotides are important. Unacceptable ○ Not capable of normal function. ○ Usually, Genetic mutation of this kind will cause severe damage to the body. ○ CAU → UAU Nonsense Mutation Convert a triple coding for an amino acid into a terminator. Premature termination of translation. Suppressor Mutation ○ Effects: protein line will be either too short it could not be secondary, tertiary, quaternary When the body mutates itself, the body reacts structures. naturally. The body creates a suppressor mutation to ○ Example: In trying to make hemoglobin then suppress a mutation. you have a premature Stop, you will have a This can counteract some of the effects of missense, very non functional hemoglobin structure nonsense, and frameshift mutation. completely. Very dangerous mutation. ○ Not all suppressor mutations are successful. Suppressor tRNA molecules, usually formed as a result of alteration in the anticodon regions, are capable of suppressing mutations ○ Sometimes they exist but they can’t stop the mutation. ○ Sometimes mutations are too strong for our body. 9  REFERENCES Buckingham, L. (2019). Molecular diagnostics: Fundamentals, Methods, and Clinical Applications. Gersen, S., & Keagle, M. B. (2010). The principles of clinical cytogenetics. Humana Press. Klug, W. S., Cummings, M. R., Spencer, C. A., Palladino, M.A., & Killian, D. (2019). Concepts of Genetics, Global Edition. Patalinhug, R (2021). DNA Replication, Transcription and Translation. Rosenberg, L. E., & Rosenberg, D. D. (2012). Human genes and genomes: Science, Health, Society. Academic Press. Weil, P. A. (2017). Protein Synthesis & the Genetic Code. Basic medical Key. Retrieved August 23, 2024, from https://basicmedicalkey.com/protein-synthesis-the-ge netic-code/ 10