Molecular Genetics: Central Dogma & DNA Replication Notes PDF

SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology Molecular Genetics Module 3: Unit 1...

SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology Molecular Genetics Module 3: Unit 1 Central Dogma and Molecular Genetics The Central Dogma explains the flow of genetic materials in organisms. The DNA can replicate and produce new DNA. It can also undergo transcription to produce RNA. The RNA undergoes translation to produce a protein. The RNA can also produce new DNA through reverse transcription. In this unit, we are going to study how genes are expressed at the molecular level. Figure 2.4.1. The Central Dogma of Molecular Biology ([DNA to RNA to Protien], nd) A. Molecular Mechanisms of DNA Replication DNA replication is important in maintaining the chromosome number per cell generation. This is to ensure that all cells in the body, except gametes, will have exactly the same genetic material. In this topic, we will learn about DNA replication in eukaryotes, then compare it with DNA replication in prokaryotes in your laboratory activity. Figure 2.4.2. Steps in DNA Replication mjmbuenaventura’24-25 1  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology 1. Initiation Recall that the DNA molecule is made up of two antiparallel DNA strands wound in a double helix. For replication to occur, the double helix must be unwound, and the DNA strands must be separated. Once separated, then the DNA can now be replicated. a. DnaB / DNA Helicase - Enzyme that binds to the DNA origin of replication initiation (ORI) sites - It unwinds and unzips the DNA forming a replication bubble with Y-shaped replication forks on each side - the replication forks serve as the initial site for DNA synthesis - eukaryotes have long, linear DNA molecules, thus would require several ORIs and form several replication bubbles Figure 2.4.3. Formation of Replication Bubbles (Samanthi, 2018) b. Topoisomerase - As the DNA splits open, the supercoil ahead of the replication fork tightens - the topoisomerase relieves the tightening of the supercoil to prevent DNA damage c. Single-stand Binding Protein (SSB) - Attaches to each separated strand, stabilizing them, and preventing them from re- annealing (going back to their original state) mjmbuenaventura’24-25 2  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology d. RNA primase - Enzyme required to specify the starting point for replication - It adds a short piece of RNA at the 3’ end of the DNA to serve as a primer - The primer will guide the enzyme for DNA replication NOTE: An RNA (ribonucleic acid) is also made up of nucleotides, but the sugar component is a ribose (DNA has deoxyribose) and one pyrimidine is Uracil (U) instead of thymine (T) Figure 2.4.4. Initiation of DNA Replication (Barton, 2016) 2. Elongation Guided by the RNA primer, the enzyme DNA Polymerase adds complementary DNA nucleotides in a 3’ to 5’ direction. Since the DNA molecule has antiparallel strands, adding nucleotides to one strand is faster than the other strand. (fig 2.4.5) Leading Strand - The 3’ end is at the opening of the replication fork, thus making it easier and faster for DNA polymerase to add DNA nucleotides - Addition of nucleotides is continuous Lagging Strand - The 3’ end is oriented on inside of the replication fork, thus making it more difficult and slower for DNA polymerase to add DNA nucleotides - Several RNA primers are required to gradually guide the DNA polymerase - Short DNA segments are formed between the RNA primers and are called Okazaki Fragments (fig 2.4.6) mjmbuenaventura’24-25 3  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology Leading strand Lagging strand Figure 2.4.5. Leading and Lagging Strands Figure 2.4.6. Formation of Okazaki Fragments DNA polymerase (alpha) is responsible for adding DNA nucleotides, using the RNA primer as a guide. After adding around 20 DNA nucleotides, elongation is taken over by DNA Polymerase ɛ (epsilon) in the leading strand, and DNA Polymerase δ (delta) in the lagging strand. DNA polymerase δ (delta) is also responsible for “proof-reading” the new DNA nucleotides that are being added in both strands. Any mistake is almost always immediately detected and corrected. 3. Termination Once all the complementary DNA nucleotides have been attached to the old DNA strands, it is time to finalize the new DNA strands. a. DNA Polymerase δ - adds DNA nucleotides to form the Okazaki Fragments, but once it encounters the RNA primer of a previous segment, it nips the bond at the 5’ end of the primer, letting it dangle as a short or long single-strand RNA, much like a flap. b. Flap Endonuclease 1 (FEN1) - Removes the daggling flap of the short RNA primer c. Dna2 Endonuclease - Removes the daggling flap of long RNA primer mjmbuenaventura’24-25 4  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology d. DNA ligase - Binds the Okazaki fragments together to form a single continuous DNA strand * * Termination is completed once the replication bubbles meet (fig 2.4.3) DNA Replication is referred to as SEMICONSERVATIVE because each DNA molecule formed is made up of an OLD DNA strand and a NEW DNA strand (fig 2.4.3) B. Molecular Mechanisms of Gene Expression The genes that are inherited by an offspring from its parents contain the instructions or blueprints for building a new organism. The use of these instructions to form an organism is what we call gene expression. The building blocks of the cell are proteins formed by one or more polypeptide chains built from a combination of amino acids. There are thousands of different types of proteins required to build an organism. The instruction for the type, number, and sequence of amino acids required to make a polypeptide for a protein is in the DNA and referred to as the genetic code. So, to make proteins, the genetic code inside the nucleus must be copied first then translated to amino acid sequences. Gene expression, therefore, has two major processes: transcription and translation. Our discussion will be concentrated on eukaryotic gene expression, then later, compare it with prokaryotic gene expression in your laboratory activity. 1. Transcription * DNA driven synthesis of RNA: the DNA is the template for the synthesis of an RNA - To transcribe means to copy, so in this process, the genetic code will have to be copied - there are three steps involved in transcription: initiation, elongation, and termination a. Initiation The DNA strand that contains the code is referred to as the sense strand while its complementary DNA strand is called the antisense strand. The antisense strand serves as the template for transcription. Figure 2.4.7. Sense and Antisense DNA Strands ❖ Transcription Initiation Complex mjmbuenaventura’24-25 5  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology A gene begins with a promoter and ends with a terminator region. The promoter contains a series of thymine (T) and adenine (A) nitrogenous bases thus called the TATA Box. The enzyme required for transcription is RNA Polymerase II. However, it cannot bind to the TATA Box on its own, thus requiring the help of several proteins collectively known as general transcription factors (TF). Since these TFs assist the RNA Polymerase II, they are all designated as TFII. TFIIs are of different types, designated as A, B, C, D, E, F, and H (there is no G). Pre-initiation Complex TFIID is the first transcription factor that binds to the TATA box because it contains a TATA Box-binding protein (TBP) - once attached, it bends the promoter by 80º, which helps in the binding of TFIIA and TFIIB TFIIA stabilizes TFIID TFIIB interacts with TBP molecule and recruits the RNA Polymerase II TFIIF assists in the binding of the RNA Polymerase II on the promoter Open Complex TFIIE binds to the pre-initiation complex and helps the binding of TFIIH TFIIH has 9 subunits, 2 of which has the ability to use ATP and acts like a helicase that splits open the promoter - the remaining 7 subunits have a kinase activity, and phosphorylates the tail of RNA Polymerase II allowing it to Figure 2.4.8. Assembly of the Transcription dissociate from the TFs, move from the Initiation Complex (Ghosh, 1996) promoter and initiate the next process called elongation b. Elongation ❖ Attachment of RNA nucleotides RNA Polymerase II facilitates the binding of complementary RNA nucleotides to the DNA Antisense strand. It forms a pre-mRNA strand that runs from the 5’ to 3’ direction. More RNA nucleotides are added until the desired code has been copied. mjmbuenaventura’24-25 6  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology Figure 2.4.9. Transcription: Addition of RNA Nucleotides by RNA Polymerase (Boundless, 2020) ❖ 5’ capping - this is the process of modifying the terminal nucleotide of the pre-mRNA RNA Triphosphatase: removes the terminal phosphate of the 5’ nucleotide Guanylyl Transferase: attaches a molecule called guanyl phosphate to the phosphate of the 5’ end forming a guanine nucleotide Methyl transferase: attaches a methyl group to the guanine nucleotide 5’ cap or Methylguanosine cap: final structure formed at the 5’ end, which protects the pre- mRNA from degradation and is also important for translation Figure 2.4.10. Formation of the Methyguanosine Cap ([mRNA’s 5’ cap structure], nd) c. Termination mjmbuenaventura’24-25 7  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology - When the RNA Polymerase reaches the terminal region of the gene, it interacts with two proteins that attach themselves to the pre-mRNA ❖ Cleaving of the pre-mRNA Cleavage Stimulations Factor (CstF): cleaves the pre-mRNA and separates it from the RNA Polymerase II - once done, CstF dissociates from the pre-mRNA ❖ 3’ Poly (A) Tail - similar to the 5’ end, the 3’ end is also protected Cleavage and Polyadenylation Specificity Factor (CPSF): still attached to the pre-mRNA and recruits Poly A Polymerase Poly A Polymerase: adds about 200 adenine(A) residues at the 3’ end of the pre-mRNA giving rise to the Poly (A) Tail Poly A binding Protein: binds to the poly A tail to prevent the degradation of the 3’ end of the pre-mRNA - CPSF is released from the Figure 2.4.11. Formation of the Poly (A) Tail pre-mRNA (Rovielli, 2015) Splicing the Pre-mRNA The synthesized pre-mRNA contains a coding sequence called exons and a non- coding sequence called introns. An enzyme called the spliceosome cuts off the introns and joins the remaining exons to form the final or mature mRNA. This mRNA is now ready to exit the nucleus and got to the cytoplasm for the process of translation. The mRNA is called a messenger RNA because it carries the message that was copied from the DNA. The copied genetic code is represented by CODONS. One codon consists of 3 nitrogen bases that code for a specific amino acid. 2. Translation mjmbuenaventura’24-25 8  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology * RNA drive synthesis of proteins: the RNA is the template for the synthesis of a protein - The genetic codes carried by the mRNA will be translated to a series of amino acids - there are three steps involved in translation: initiation, elongation, and termination a. Initiation Three types of RNAs participate in the process of translation. The mRNA (messenger RNA) that carries the codons, the rRNA (ribosomal RNA) that combines with proteins to form ribosomal subunits, the tRNA (transfer RNA) with an amino acid binding site on one end, and an anticodon on the other end. The anticodon is a complement of the codon carried by the mRNA. Figure 2.4.12. Types of Ribonucleic Acids for Translation ([messenger RNA], nd) The small subunit and large subunit of the ribosome have three identified sites. The A- site (Aminoacyl Site) for attachment of tRNA carrying an amino acid. The P-site (peptidyl site) where tRNA with an amino acid forms a peptide bond to form an amino acid chain. The E-site (exit site) is where tRNA with no amino acid exits the ribosome. Figure 2.4.13. Sites of the Ribosome (Shah, 2015) mjmbuenaventura’24-25 9  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology ❖ Eukaryote Initiation Factors (eIFs) eIF-1, eIF-1A, and eIF-3: bind to small subunit of ribosome eIF-2: brings the initiator methionyl tRNA (tRNA-met) to the small ribosomal subunit at the P-site - tRNA is called an initiator because is the first tRNA to start translation - it is also called methionyl because it is carrying the amino acid methionine, which is coded by the start codon AUG eIF-4E: binds to the 5’ cap of the mRNA eIF-4G: binds to the poly (A) tail eIF-4A and eIF-4B: bring the mRNA (with attached eIF-4E and eIF-4G) to the small ribosomal subunit - the ribosome scans the mRNA to look for the initial or start codon AUG - once identified, the last type of eIF goes to work eIF-5: triggers the release of eIF-2 from tRNA-met and all other eIFs detach - signals the large ribosomal subunit to complex with the small subunit Figure 2.4.14. Eukaryotic Initiation of Translation (Cooper, 2000) mjmbuenaventura’24-25 10  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology b. Elongation ❖ The Genetic Code - The start codon AUG codes for the amino acid methionine, and that was the amino acid that initiator tRNA picked up - the next amino acid to be picked-up by tRNA depends on the second codon - below is the table for the genetic code showing the corresponding amino acid of specific codons FIG. 2.4.15 The Genetic Code ❖ tRNA Delivers Amino Acids Aminoacyl synthase: an enzyme that binds the amino acid to the tRNA - tRNA brings the amino acid to the A-site of the ribosome - the tRNA anticodon complexes with the mRNA codon ❖ Transpeptidation Peptidyl Transferase: catalyzes the binding of the second amino acid to the first (methionine) amino acid by forming a peptide bond ❖ Translocation - the ribosome slides towards the next codon as it frees the A-site - tRNA with no amino acid exits via the E-site mjmbuenaventura’24-25 11  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology The next amino acid is picked up by another tRNA, tRNA delivers the amino acid, transpeptidation occurs, translocation occurs, and the process is repeated over and over as the amino acid chain grows. Figure 2.4.16. Eukaryotic Elongation during Translation (Harrison, 2019) c. Termination When the ribosome reaches the stop codon (UAG, UAA, UAG), it signifies translation termination. There is NO anticodon for the stop codon, thus no tRNA is involved during termination. However, a cytoplasmic termination factor (CTF) or release factor (RF) binds with the stop codon, signaling the peptidyl transferase to transfer the amino acid chain to a molecule of water. Once done, the ribosomal subunits detach and the mRNA nucleotides separate, to be used again for the next transcription process. mjmbuenaventura’24-25 12  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology Figure 2.4.17. Eukaryotic Termination of Translation (Cedarbaum, 2015) 3. Reverse Transcription Reverse transcription is used by retrovirus to express their genes inside hosts cells. It is an RNA-driven DNA synthesis. The viral RNA is used as a template to synthesize a single-strand DNA, which in turn will be used to synthesize the complementary DNA strand, forming a new DNA double-stranded molecule. This new DNA will then be used to synthesize numerous copies of viral RNA as well as proteins for forming viral envelopes and surface markers. mjmbuenaventura’24-25 13  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology mjmbuenaventura’24-25 14  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology Figure 2.4.1. Retrovirus Reverse Transcription (MoBio, nd) Steps in Retroviral Reverse Transcription (fig 2.4.16) (1) tRNA primer (pink) binds to the primer binding site (PBS) of the viral RNA (red) (2) Reverse transcriptase (RT) is an enzyme that uses the primer as a guide and adds complementary DNA nucleotides to the viral RNA, forming a short segment DNA (ss DNA) (green) (3) RNAse H is an enzyme that degrades the copied viral RNA (red R and U5) (4) RT, ssDNA, and tRNA detach and undergoes the first jump to the other end of the viral RNA (5) RT adds more DNA nucleotide to complete the first DNA strand (green) (6) RNAse H degrades the copied viral RNA except in a region called the polypurine tract (red) mjmbuenaventura’24-25 15  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology (7) RT uses the polypurine tract as a guide to add complementary DNA nucleotides (short green) to the first DNA strand (long green) (8) RNAse H degrades the polypurine tract (9) The second short segment DNA undergoes a second jump and its PBS complements with the PBS of the first DNA strand in some retroviruses, instead of undergoing a second jump, the entire DNA in fig (8) undergoes circularization so that the PBS of both DNAs would bind (fig 2.4.17) Figure 2.4.19. Circularization of the Viral DNA strand (Iwatani, 2007) (10) RT completes the second DNA strand, forming a double-stranded viral DNA with antiparallel strands This DNA can now be used as a template to: transcribe numerous copies of viral RNA (this is how they reproduce and increase in number) transcribe mRNA for translation to proteins that form viral envelopes Figure 2.4.20. Structure of a and enzymes Retrovirus (California Academy of Sciences, 2016) mjmbuenaventura’24-25 16  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology The process of reverse transcription is now being capitalized in the production of DNA copies (cDNA) in laboratories, a process called polymerase chain reaction (PCR). Recall the S-phase of cell division. This is the stage where the DNA is replicated before undergoing division. In PCR, numerous DNA strands are formed even without cell division. This is called DNA amplification. C. Molecular Mechanism of Gene Regulation All cells in the body contain a complete set of genes but are not expressed at the same time. Gene expression is in a precise and regulated fashion, depending on the type of cell. Genes do not only code for structural proteins. They also code for functional proteins called enzymes. Enzymes are molecules that catalyze metabolic processes inside the cell. They are especially important for the normal functioning of the cells. However, not all enzymes are synthesized or activated simultaneously. They are synthesized only when required, so as not to wreak havoc inside the cell. How does a cell know when to activate or inactivate a gene? How do cells regulate gene expression? Prokaryotic Gene Regulation In 1961, Francois Jacob and Jacques Monod proposed the Operon Hypothesis for gene regulation in prokaryotes. According to the hypothesis, prokaryotes have a system of genes called OPERON, that regulates gene expression. It of made up of several adjacent structural genes (cistrons) that code for required proteins. Beside the cistrons is the operator that controls transcription. Adjacent to the operator is the promoter for RNA polymerase binding. Elsewhere in the DNA is a regulatory gene that codes for a regulatory protein in charge of switching the operator on or off. Figure 2.4. 21. Components of an Operon System ([Prokaryote structural genes], nd) mjmbuenaventura’24-25 17  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology 1. Inducer Operon System * Lactose (Lac) Operon - the lactose operon system is a type of inducer operon system that Jacob and Monod discovered Lac Operon Components a. Lac Cistrons The Lac Operon System has three cistrons, thus the mRNA produced during transcription is called Polycistronic (transcribed from several genes or cistrons) o Lac Z transcribes for galactosidase o Lac Y transcribes for lactose permease o Lac A transcribes for transacetylase b. Operator lies adjacent to the Lac Z cistron in the absence of a substrate (lactose) to breakdown, no enzyme is required by the cell, thus transcription is not needed a repressor protein binds to the operator to prevent transcription c. Promoter located adjacent to the operator for the binding of RNA polymerase the base sequence of the promoter determines which DNA strand will serve as the template for transcription d. Lac Repressor - a type of regulatory protein synthesized by the regulatory gene - it binds to the operator gene to turn it off f. Lac Inducer - a molecule that binds to the repressor protein to inactivate it Lac Regulatory Steps a. Binding of Lac Repressor - in the absence of a substrate (lactose) to breakdown, the Lac repressor (regulatory protein) binds to the operator preventing transcription to occur - once bound, the DNA polymerase that is attached to the promoter cannot pass through the operator to transcribe the structural genes b. Formation of Lac Inducer - a small amount of lactose permease is present in the cell (E. coli) mjmbuenaventura’24-25 18  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology - when lactose enters the cell, the lactose permease breaks down some of the molecules - the lactose molecules are then converted into an active form to become a Lac inducer c. Binding of Lac Inducer - The Lac inducer binds to the repressor causing it to detach from the operator - once removed, the DNA polymerase that is attached to the promoter can now pass through the operator and promote transcription - as long as the inducer is attached to the repressor protein, the repressor cannot bind to the operator the lac molecule is called inducer because it allows transcription to occur by inactivating the repressor d. Transcription - DNA Polymerase binds ( or bound) to the promoter gene and promotes transcription of galactosidase, lactose permease, and transacetylase for the breakdown of all the lactose that has entered the cell e. Release of Repressor - When all the lactose has been broken down by the enzymes and used by the cell for energy production, there will be no more Lac inducer left to bind to the Lac repressor - The Lac repressor can once again bind to the operator, switching it off, and preventing transcription to occur - This is to ensure that no more enzyme would be produced since there is no more lactose to breakdown Figure 2.4.22. Lactose Operon System ([five structural genes needed to synthesize tryptophan in E.coli], nd) mjmbuenaventura’24-25 19  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology 2. Repressor Operon System * Tryptophan (Trp) Operon - the tryptophan operon system is a type of repressor operon system Trp Regulatory Components a. Trp Cistrons The Trp Operon System has five cistrons, thus the mRNA produced during transcription is also called Polycistronic o Trp E and Trp D transcribes for anthranilate synthase o Trp C transcribes for indoglycerol phosphate synthase o Trp B and Trp A transcribes for tryptophan synthase b. Operator lies adjacent to Trp E it remains turned on because a regulatory protein is not bound to it so, there is a continuous synthesis of enzymes to catalyze the synthesis of tryptophan from raw materials c. Promoter located adjacent to the operator for the binding of RNA polymerase the base sequence of the promoter determines which DNA strand will serve as the template for transcription e. Aporepressor Protein - regulatory protein synthesized by the regulatory genes - cannot bind to the operator on its own thus needs a corepressor f. Corepressor - a non-protein compound that may either come from outside of the cell or a product of metabolism within the cell Regulatory Steps a. Aporepressor-corepressor Complex - The cell is continuously producing tryptophan - however, if tryptophan somehow accumulates, or additional tryptophan is received from outside of the cell, further synthesis must be stopped - the tryptophan itself selves as the co-repressor and binds to the aporepressor, forming the aporepressor-corepressor complex. b. Binding to the Operator mjmbuenaventura’24-25 20  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology - the aporepressor-corepressor complex binds to the operator, preventing the RNA polymerase from passing through, thus the synthesis of enzymes is stopped Tryptophan is called repressor because it stops transcription c. Release of the Aporepressor - When all the levels of tryptophan are decreased, the corepressor releases the aporepressor causing it to detach from the operator - DNA polymerase can once again pass through the operator and promote the synthesis of enzymes for tryptophan production Figure 2.4. 23. Tryptophan Operon System Eukaryotic Gene Regulation Gene expression in eukaryotes depends on external cues as cells grow and develop. Gene regulation, therefore, helps the cells (and eventually the entire organism) to cope with their changing environment. This is the molecular basis of the growth and development of organisms. Unlike prokaryotes, the genes required for the synthesis of enzymes are not adjacent to each other. They may be located farther from each other on the same DNA or may even be located in different DNAs. Like prokaryotes, there are also inducible and repressible systems but involve a complex of regulatory genes. In addition, gene expression occurs at different mjmbuenaventura’24-25 21  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology stages of transcription and translation. 1. Regulation of Transcription Factors - transcription factors (TF) are molecules that bind to promoters to initiate transcription a. Regulation of Nuclear Localization - when not required in the nucleus, transcription factors stay in the cytoplasm - when required, they are phosphorylated and enter the nucleus - this regulation ensures that only required transcription factors enter the nucleus and bind to their respective promoter b. Regulation of DNA-binding - even if TFs are already inside the nucleus, DNA-binding is still regulated in two ways Alteration of the DNA-binding domain - the DNA-binding domain is a portion of the TF that binds to the DNA - it must first undergo a conformational change in the domain so that the TF can finally bind to the promoter Multimerization - some TFs need to bind with other TFs forming a complex first to become active and eventually be able to bind to the promoter 2. Regulation of Transcription - assuming that the transcription factors were able to bind with the promoter, transcription will still be regulated by either activators or repressors a. Activators - turn on transcription - activators have two domains, a DNA-binding domain, and an activation domain - activation may either be a short distance or long distance Figure 2.4.24. Short-distance and Long-distance Activation of Transcription (Kulaeva, 2012) mjmbuenaventura’24-25 22  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology Short distance activation - DNA-binding domain binds to an enhancer DNA located beside the promoter - the activation domain facilitates the assembly and binding of the transcription factor complex with RNA Polymerase (RNAP) on the promoter - once the TF complex is attached and activated, transcription may proceed Long-distance activation - DNA-binding domain binds to an enhancer DNA located far from the promoter - the DNA undergoes looping bringing the activator close to the transcription factor complex for activation - once the TF complex are activated, transcription may proceed b. Repressors - turn off transcription - these are proteins that bind to specific sites of the DNA to prevent transcription of nearby genes - transcription inhibition may done in either of these three ways Repressor binds to promoter to prevent the transcription factor complex from binding Repressor binds to enhancer to prevent the activator from binding Repressor binds to a silencer gene with its DNA-binding domain - Binding causes the DNA to undergo looping, bringing the repression domain close to the promoter 3. Regulation AFTER Transcription - transcription may have occurred but another mechanism can regulate gene expression after transcription a. Regulation of mRNA Processing Figure 2.4.25. Activity of Transcription Repressors - one pre-mRNA can produce (Microbial Genetics, nd) different types of final mRNA mjmbuenaventura’24-25 23  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology - regulatory proteins bind to the mRNA and tell the spliceosomes where to cut the mRNA Different exon combinations will contain different codons, thus translating for different polypeptides Figure 2.4.26. Differential mRNA Splicing (Di, 2018) b. Regulation by miRNAs - micro RNAs (miRNAs) are non-coding RNAs consisting of 20 to 24 RNA nucleotides - they bind to mRNAs that have not been translated for quite sometime - basically, miRNAs are responsible for the life span of mRNAs If the RNA nucleotide sequence is complementary to a segment of mRNA, then the mRNA is chopped and will no longer be translated (discarded) If the RNA nucleotide sequence is only partially complementary to any mRNA segment, the binding simply prevents translation mjmbuenaventura’24-25 24  SAINT LOUIS UNIVERSITY SCHOOL OF NURSING, ALLIED HEALTH, AND BIOLOGICAL SCIENCES Department of Biology 4. Regulation of Translation - if the cell cannot “afford” to make new polypeptides and proteins due to the lack of nutrients or raw materials, particularly amino acids (even if there is a signal from growth factors), translation is inhibited - eIFs that participate in the translation are phosphorylated and rendered inactive, thus translation cannot occur 5. Regulation AFTER Translation - even if the translation has occurred, there is still one last mechanism for regulation of gene expression a. Phosphorylation - either activates or deactivates the newly produced protein b. Ubiquitination - ubiquitin binds to proteins and delivers them to the proteasome for degradation - the amino acids released from degradation will be recycled for the next translation - this process controls the persistence of proteins in the cell - if the proteins have been synthesized but are not yet used for quite some time, ubiquitination occurs NOTE: Facultative Genes are genes may be turned on or off depending on the need of the cell. Constitutive Genes (housekeeping genes) are never turned off because they are important for the maintenance of the cell. 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