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Chapter 8. Central Dogma

Higher category: 【Biology】 Biology Index 

1. Central Theory

2. Genes and Gene Expression

3. DNA replication

4. Transcription

5. Translation

6. Delivery of Proteins After Translation

7. Post-translational Modification (PTM)

8. Inhibitor

1. Overview of central dogma 

⑴ Central theory refers to the flow of genetic information (DNA → RNA → protein)

⑵ Revised central theory after the reverse transcriptase is known in retroviruses (RNA viruses)

⑶ Information transfer relationship

① DNA replication: DNA → DNA

② Transcription: DNA → RNA

③ Translation: RNA → amino acid sequence (primary structure)

④ Triplet code (DNA) → codon (mRNA) → anticodon (tRNA) → amino acid

2. Genes and Gene Expression

⑴ Genes and Chromosomes

⑵ 1 gene multi polypeptide description

① 1 gene 1 enzyme: Beadle and Tatum’s experiment

○ Inferring Biosynthetic Pathways of Materials Using Mutants: Ornithine, Citrulline, Arginine

Table 1. Beads and Tatum Experiments

○ Know-how 1. The more growing the material, the later stages of the biosynthetic pathway.

○ Know-how 2. The more the mutant grows, the more involved it is in metabolism

○ Choose only know-how 1 or know-how 2 to solve the problem!

② 1 gene 1 protein theory: Proteins that do not function as enzymes are found

③ 1 gene 1 polypeptide theory: Proteins Forming Quaternary Structures Revealed

④ 1 gene multi polypeptide theory: Cases where multiple polypeptides are produced from one gene

⑶ Regulation of Gene Expression

① Overview of gene expression

○ First, the somatic cells of our body have the same DNA

○ Second, each tissue cell only transfers specific genes, resulting in different gene expression patterns, resulting in different structures and functions.

② Transcription

○ Regulation by transcriptional inhibition (prokaryotic cells)

○ Regulation by transcriptional activation (prokaryotic, eukaryotic)

○ Control by Chromosomal Condensation: Access prevention of RNA polymerase (Baso body)

③ Regulation by mRNA Degradation

④ Control of translation

⑤ Regulation of Proteolysis (Protease)

3. DNA replication

⑴ 1^st. Yawning DNA helix

① Replication proceeds in both directions from the replication origin (ori) (= the replication fork moves in both directions from the replication origin)

○ Replication origin: A = T rich site with weak hydrogen bonds

○ Replication Junction: The junction between the strand where the DNA is hydrogen-bonded and the strand that is not

○ Prokaryotes: DnaA recognizes one copy origin

○ DnaA: consensus; 9 bases × 4 + 13 bases × 3

○ Eukaryotes: ORC (origin recognition complex) recognizes origin of replication

○ Multiple replication origins due to slow replication

○ Leaven: 250 to 400 replication origins

○ Mammal: 25,000 replica points

○ ARS (autonomic replicatoin sequence)

○ DnaA and ORC generate replication bubbles that expose tens of nts of single strand DNA (ssDNA)

② Enzyme: Helicase (DnaB), DNA topoisomerase, single chain binding protein

○ Helicase: Releases hydrogen bonds in two complementary strands

○ Helicase characteristic experiment: Conclusion 1, conclusion 2 can be obtained

○ Conclusion 1. Binding to DNA requires DnaA or ORC-generated replication

○ Conclusion 2. Typically binds to the delayed strand template at the DNA replication junction. 5 ‘→ Go to 3’

○ Enable ATP. Minimum ssDNA length of replica bubble: 16 nucleotides or more

○ The helicase acts to intensify the DNA’s torsion, resulting in unstable structure

Figure 1. Topological isomer of circular DNA of 210 bp in size

The second double helix has less nt per revolution (small load). 3rd double helix has more nt per revolution (large load)

○ L: Rings. The amount that is preserved when calculating the rotational load.

○ T: Revolutions. DNA is basically helical and has a counterclockwise rotational load

○ W: Supercoil

○ supercoil (supercoil): DNA molecules themselves ringing. Related to strengthening and mitigating rotating loads

○ Benign supercoil: Left side supercoil (third in the figure above). supercoil twist is positive

○ Negative supercoil: Right side supercoil (second in figure above). supercoil twist is negative

○ Intuitive understanding. The positive supercoil draws a supercoil in the left helix and thus increases the rotational load in the right helix of the double helix.

○ Intuitive understanding. Negative supercoil relieves the rotational load in the right helix of the double helix while drawing the supercoil in the right helix

○ Most organisms that are not thermophilic bacteria have DNA with a negative supercoil structure

○ DNA spine: Enzymes to relax the rotational load of DNA

○ Acting in front of distant point at replication

○ DNA holoenzyme Ⅰ: An enzyme that breaks one point of the DNA strand, loosens it naturally, and then recombines

○ DNA holoenzyme Ⅱ (DNA gyrase): Breaks two phases of the DNA strand, turns (phase changes) and recombines an enzyme, a DNA molecule that was a positive supercoil

○ Single strand binding protein (SSBP): Temporarily binds each unstranded DNA strand to prevent single-stranded pairs again. Proteins That Stabilize DNA

○ Experimental SSBP is required to activate the action of the helicase

③ Prokaryotic cells: Circular DNA, one replication origin

④ Eukaryotic cells: Linear DNA, multiple replication origins

○ Eukaryotic genomes are larger than prokaryotic cells and have many origins of replication..

⑵ 2^nd. Attaching primer

① Primer: Short RNA Fragments Underlying DNA Polymerization

○ PCR primers are DNA fragments.

② Primers provide the first 3’-OH group ends needed for DNA polymerization

③ Enzyme: Primase is involved

⑶ 3^rd. DNA polymerization

① Enzyme: DNA polymerase is involved

○ E. coli DNA polymerase’s DNA polymerase enzymes include DNA pol I, II, and III.

○ DNA pol I: Nick translation. Removes primers and replaces RNA primers with dNTP. Speed: 10 nt/s.

○ T4 ligase performs functions similar to the DNA pol I subunit.

○ DNA pol II: Activated when DNA is damaged by external factors, involved in DNA repair, and activated when DNA synthesis is interrupted.

○ DNA pol III: Acts as a DNA elongation and replicative polymerase, with high potency, fidelity, and processivity. Speed: 1000 nt/s. Shows asymmetry in elongating the lagging and leading strands.

○ Characteristics: Very high potency, fidelity, and processivity.

○ DNA pol III is composed of about 10 different polypeptide chains.

○ Overall structure: αεθβ₂τ₂ - αεθβ₂γ₂(δδ’χψ)₂.

○ α subunit: Polymerase.

○ ε subunit: 3’ → 5’ exonuclease.

○ β₂τ₂ : Processivity, star-shaped ring, 35 Å hole.

○ β₂: Sliding DNA clamp-related.

○ Sliding clamp

Figure 2. Sliding clamp

○ Maintains proper alignment of the template and primer strands for successful DNA synthesis—increases accuracy and efficiency.

○ In prokaryotes, the sliding clamp involves PCNA (proliferating cell nuclear antigen) in eukaryotes and is associated with Ki-67 staining.

○ In eukaryotes, the sliding clamp is temporarily opened by the clamp loader, allowing insertion of the closed circular structure of E. coli DNA into the central channel. It facilitates the activity of DNA polymerase during DNA replication.

○ Klenow fragment: A portion of DNA pol I involved in 5’ → 3’ polymerization, 3’ → 5’ exonuclease, and 5’ → 3’ exonuclease activities.

○ (Note) DNA pol I: 5’ → 3’ polymerization, 3’ → 5’ exonuclease, 5’ → 3’ exonuclease.

○ (Note) DNA pol I large fragment: 5’ → 3’ polymerization, 3’ → 5’ exonuclease.

○ (Note) DNA pol I small fragment: 5’ → 3’ exonuclease.

○ (Note) 5’ → 3’ exonuclease: Terminal nucleotide removal function.

○ (Note) 3’ → 5’ exonuclease: Proofreading function.

○ DNA pol I’s large fragment has the same functions as DNA pol III.

○ T4 polymerase’s DNA polymerase.

○ Restriction enzyme sites in DNA recombination change sticky ends to non-sticky ends.

○ 5’ → 3’ exonuclease activity is absent, preventing the degradation of the leading DNA strand’s primer.

○ Example of in vitro polymerization using the Klenow fragment

Figure 3. Example of in vitro polymerization using the Klenow fragment

○ In eukaryotes, DNA pol α, β, γ, δ, and ε exist.

○ DNA pol α: Contains primase, lacks proofreading.

○ DNA pol β: Repair function, lacks proofreading.

○ DNA pol δ: Extends the Okazaki fragments of the lagging strand, proofreading.

○ DNA pol ε: Extends the leading strand, proofreading.

○ DNA pol γ: Replicates mitochondrial DNA, proofreading.

② Substrate: dNTP (dATP, dTTP, dCTP, dGTP), with 3 phosphate groups on 5 ‘carbon

③ Release 2 pyrophosphates by attaching the 5 ‘phosphate group of nucleotides complementary to the template to the 3’-OH group of the synthetic strand

○ 3’-OH group in DNA double helix structure accurately attack α phosphate group

④ A is complementary to T and G to C (the distance between the two strands of DNA, purine-pyrimidine is always constant)

⑤ Composite direction: 5 ‘→ 3’

⑥ Leading strand

○ Subsequently compounded chain

○ When the direction of replication is the same as the direction of movement of the replication branch

⑦ Lagging strand

○ Chains synthesized into discrete segments (Okazaki slices)

○ When the direction of duplication and the direction of duplication are reversed

⑷ 4^th. Chain closing

① Termination mechanisms exist but are not important because they replicate 100% by default

⑸ 5^th. Primer Removal

① RNA primers are removed by DNA polymerase I

② When the RNA primer is removed, DNA ligase (ligase, ligase, DnaG) performs DNA chain linkage reaction

○ Legacy uses ATP

○ DnaB and DnaG are collectively called primosomes

③ RNA primer at the 5 ‘end of the daughter chain could not be replicated → DNA length becomes shorter with DNA replication

○ Related to aging

⑹ 6^th. Correction and Modification of DNA Replication: Exonuclease activity

① Original marker: Methylated base A of template strand to indicate originality

○ DNA methylase is involved. Methylation of 5’-GATC-3 ‘Sequence at the ori C Site

② Correction: DNA polymerases have correction. Replication error rate during polymerization is 1/107

○ Exonuclease

○ 3 ‘→ 5’ modification (DNA pol I, II, III): 3 ‘→ 5’ exonuclease activity allows correcting errors in DNA synthesis

○ 5 ‘→ 3’ modification (DNA pol I): 5 ‘→ 3’ exonuclease activity

③ Repair: If the repair enzyme corrects, the error rate of replication is 1/109.

○ Dimer Recovery

○ Resection: Abnormal base removal caused by chemical damage

④ Mutations continue when DNA repair and repair fail

⑺ Characteristics of DNA Replication

① Semi-conservative replication: After replication, there is one template strand and one replication strand in each DNA

○ Conservative Replication: Existing DNA and New DNA exist after each copy

○ Distributed replication: Two completely new DNAs are created after cloning

○ Meselson-Stahl’s experiment

Figure 4. Meselson-Stahl’s experiment

○ Density gradient centrifugation using cesium chloride → DNA separation with different densities

○ Escherichia coli labeled with 15N was analyzed for DNA density every generation in medium containing normal nitrogen (14N).

② Complementarity: Template strand and clone strand have complementary information

⑻ Telomerase: 2009 Nobel Prize in Physiology or Medicine

① RNA primer moiety at 5 ‘end of daughter chain could not be replicated → DNA shortened → Aging

○ Hayflick limit: The number of splits until you can no longer split

② telomere (telomere)

○ Protect to some extent from shortening of DNA

○ Meaningless repetition, TTAGGG for humans, approximately 300 to 5000 repetitions

Figure 5. Mechanism of Exposed 3’ Terminus in Telomeres and Telomerase

③ Teller Race: Reverse Transcriptase, RNA Dependent DNA Polymerase

○ Short RNA in molecule lengthens telomere again

○ Short RNA in the molecule: 3’-CCCAAUCCC-5 ‘RNA template

④ Found in germ cells, proliferating normal cells, cancer cells, circular DNA (eg: mitochondria DNA) does not require telomerase

⑤ Tetra G on telomere forms a t-loop, transforming the exposed telomere 3 ‘end into a hairpin structure to protect the end

⑼ RCR(rolling-circle replication): Only observed in circular DNA.

Figure 6. rolling-circle replication

⑽ multi-fork replication

In an extremely good environment, the breeding cycle of E. coli is 20 minutes. The replication rate of DNA is 1 second per 1000 nucleotides, so it takes longer than the reproduction cycle to replicate all the chromosomes. Nevertheless the reason why E. coli has a short breeding cycle is because of multi-fork replication. This is a phenomenon in which replication begins on a daughter strand that is already in progress, preparing for the next cell cycle before one cell cycle is completed..

4. Transcription

Figure 7. Gene transcription</center>

⑴ DNA vs. RNA

① Strands: DNA is double stranded, RNA is single stranded

② Party: DNA is deoxyribose (pentose), RNA is ribose (pentose)

○ Deoxyribose has -2 carbon in -H group, ribose has -2 carbon in -OH group

③ Base: A, T, G, C (DNA) ↔ A, U, G, C (RNA)

④ DNA type

○ gDNA (Genomic DNA): Typical DNA.

○ Mobile DNA

○ Satellite DNA: Non-coding DNA that randomly repeats within eukaryotic genomes.

○ Plasmid: Only found in bacteria

○ ecDNA (Extrachromosomal DNA): Circular DNA that exists outside of chromosomes, contributing to high copy numbers of specific genes in cancer cells.

⑤ RNA type

○ mRNA (messenger RNA, messenger RNA)

○ tRNA (transfer RNA)

○ rRNA (ribosome RNA, ribosome RNA)

○ SRP RNA

○ SnRNA

○ miRNA, siRNA

○ lncRNA and ncRNA: Non-coding RNA (e.g., pseudo genes are transcribed but not translated)

○ sciRNA

○ ASO(antisense oligonucleotide)

○ snoRNA (small nucleolar RNA)

○ glycoRNA

⑵ Prokaryotic and Eukaryotic mRNAs

① Polycistron, monocistron

○ Prokaryotes: Polycystron (= operon)

○ When one mRNA molecule encodes several different polypeptide chains

○ The starting region of each polypeptide has a ribosome binding site (RBS)

○ Advantages of controlling the expression of several genes with one promoter

○ Polyribosome (polysome): Multiple ribosomes bound to one mRNA

○ Eukaryotes: Monocystron

○ Polyribosomes are also observed in eukaryotes, but in very small quantities

○ RRNA: Polyribosomes are also observed in eukaryotes

② presence of intron: Has only eukaryotes

○ Histone DNA: no intron

③ Concurrency of transcription and translation: Only prokaryotes progress simultaneously

④ Post Transcription Deformation Process: Only eukaryotes exist

⑶ 1^st. RNA chain initiation: RNA polymerase binds to gene promoter

① RNA releases closed promoter complexes into open promoter complexes

② Both strands of the DNA chain encode genes, but any single gene uses only one strand of DNA as a template

○ In the narrow range, only one strand of DNA can be considered as a template

○ sense strand (password strand, (+) strand, non-template strand): RNA polymerase free chain, easy for codon analysis

○ anti-sense strands (non-coding strands, (-) strands, template strands): RNA polymerase chain

○ Example: Yeast galactose genes (6 total)

○ 4 on chromosome 2 (GAL7, GAL10, GAL1, MEL1), 1 on chromosome 12 (GAL2), 1 on chromosome 4 (GAL3)

○ GAL7, GAL10, GAL1 are on one strand, MEL1 is on the other strand

③ Promoter

○ About 40 base pairs of DNA indicating transcription initiation site

○ Site where RNA polymerase binds

○ Consensus (consensus) of prokaryotic promoters

○ Prokaryotes have one promoter

○ Example 1. Pribnow box (-10 box): TATAAT

○ Example 2. -35 box: TTGACA

○ Eukaryotic promoter consensus (consensus)

○ Eukaryotes have three promoters

○ Example 1. TATA box (Goldberg-Hogness box, -25 box): TATA

○ Example 2. CAAT box (-75 box): CAAT

○ Example 3. GC box

④ Upstream / downstream

○ First nucleotide to be transcribed into RNA is called +1

○ 5 ‘side upstream (-) and 3’ side downstream (+)

⑷ 2^nd. Elongation of the RNA chain: RNA polymerase polymerizes RNA base complementary to DNA base (template strand) of gene into 5 ‘→ 3’ to produce RNA as a product of transcription

① transcription bubble = RNA transcript + DNA + RNA polymerase

② RNA-DNA hybrid

③ RNA polymerase: No primer required, no topoisomerase, no exonuclease activity, transcription error rate ↑

④ E. coli DNA dependent RNA polymerase

○ α2ββ’ω: Binding to a promoter with the help of the σ70 factor of recognition

○ σ70: Recycle

⑤ Eukaryotic DNA dependent RNA polymerase

○ RNA pol I: Action on phosphorus, 28S rRNA, 18S rRNA, 5.8S rRNA Synthesis

○ RNA pol II: Action in the nucleus, synthesis of mRNA

○ RNA pol III: Action in the nucleus, tRNA, 5S rRNA, ncRNA, snRNA synthesis

⑸ 3^rd. Termination of the RNA chain: In the terminator sequence, RNA polymerase leaves DNA and DNA twists again

① Prokaryotic terminator

○ An implicit terminator: Termination of transcription by forming hairpin-oligo-U structures

○ Hairpins are formed by a bivalent symmetric sequence

○ A-T rich site located not far from bivalent symmetric sequence transfers U base using A as template

○ Unstable bonds in A = U bond chains, which naturally separate transcripts

○ Rho-dependent terminator: Rho factor acts as a DNA-RNA helicase to promote the escape of RNA during synthesis

② Eukaryotic Terminator

○ Endonuclease Activity Following Poly A Formation Signal Sequence (5’-AAUAAA-3 ‘) of Transcript

○ Endonuclease breaks down after cutting 10-35 degrees

○ Poly A tail formation with multiple A bases linked at the truncated 3 ′ end

⑹ 4^th. mRNA processing (eukaryotic cells only): Mature mRNA is transferred to the cytoplasm through nuclear pores, degrades and translates

① 1st. capping

Figure 8. 5’-cap

○ Transcription begins simultaneously.

○ 7-MeG, a methylated guanosine derivative, is linked to the 5’ end of pre-mRNA via a 5’-5’ bond (5’ carbon - 3 phosphate groups - 5’ carbon).

○ The 5’-MeG binds to CBC (cap binding complex) to form a 5’-cap, also known as m7GpppN.

○ Cap: Helps nuclear export, protects mRNA from nucleases, and serves as a recognition site for protein synthesis machinery.

○ Artificial 5’-cap for IVT (in vitro transcription):

○ 1st generation: mCap (Cap 0) derived from yeast; lacks directionality.

○ 2nd generation: ARCA (anti-reverse cap analog) (Cap 0 mimic) derived from yeast; introduces a methyl group for directionality.

○ 3rd generation: Trinucleotide cap analog (e.g., CleanCap) (Cap 1), most similar to natural 5’-cap and superior.

② 2nd. Poly A tail formation

○ Poly A tail is related to transcription terminators in eukaryotes.

○ PBP (poly A binding protein) adds A bases following the 5’-AAUAAA-3’ sequence.

○ Poly A tail aids mRNA transport from the nucleus to the cytoplasm and increases mRNA stability.

③ 3rd. Splicing

○ Performed by spliceosomes, connecting exon regions only. Requires ATP.

○ Exons: Encode functional genetic information and untranslated regions (UTRs) for translational regulation.

○ Introns: Encode non-functional information.

○ May exist to maintain distances between DNA in chromatin.

○ Speculated to have deeper significance.

○ Spliceosome (snRNP): Composed of snRNA.

○ u1 snRNP + u2 snRNP + u4 snRNP + u5 snRNP + u6 snRNP → Removal of u1 snRNP and u4 snRNP.

○ Conserved sequences in pre-mRNA for splicing:

○ Splicing acceptor: GU at the 5’ splice site and AG at the 3’ splice site.

○ 5’-GU—AG-3’, pyrimidine-rich site, branch point A.

○ Group I self-splicing

Figure 9. group I self-splicing

○ Step 1: External guanosine molecule attacks the 5’ splice site, binding to the intron and leaving the 5’ exon with a 3’ OH group.

○ Step 2: The 3’ end of the 5’ exon attacks the 3’ splice site, joining the 5’ exon to the 3’ exon and excising the intron.

○ Group II self-splicing

Figure 10. group II self-splicing

○ Step 1: Internal adenosine molecule attacks the 5’ splice site, forming a lariat structure.

○ Step 2: The 3’ end of the 5’ exon attacks the 3’ splice site, joining the 5’ exon to the 3’ exon and excising the intron.

○ Difference 1. ** **Initial nucleophile: External guanosine in Group I vs. internal adenosine in Group II.

○ Difference 2. Structure: Complex loops and helices in Group I vs. lariat in Group II.

○ Difference 3. Distribution:

○ Group I: rRNA, mRNA, tRNA in bacteria; mitochondrial/chloroplast genomes in lower eukaryotes; some tRNA/mRNA in higher plants.

○ Group II: rRNA, tRNA, mRNA in organelles of fungi, plants, protists; bacterial mRNA.

○ Summary: Group I is more common in lower organisms (e.g., bacteria), and Group II in higher organisms (e.g., fungi, plants).

④ 4^th. mRNA editing (RNA editing)

○ Rare compared to capping, poly A tail formation, and splicing.

○ Guide RNA serves as a template.

○ Type 1: Protein length remains unchanged.

○ Type 2: Protein length changes (e.g., apolipoprotein B protein).

○ Apo-B100 (100 kDa) in the liver.

○ Apo-B48 (48 kDa) in the small intestine.

○ Cytidine deaminase deaminates 5’-CAG-3’ to 5’-UAG-3’, creating a new stop codon.

⑤ 5th. Alternative splicing

○ Some exons on a single pre-mRNA are selectively included, forming multiple mRNA combinations.

○ >95% of intron-containing human genes undergo alternative splicing.

○ Result 1. Improper splicing removes normal exons → May cause cancer.

○ Result 2. Premature stop codons → Degradation of abnormal mRNA by NMD (nonsense-mediated mRNA decay).

○ Alternative splicing events:

Figure 11. Types of mRNA splicing

○ SE (Skipped Event): An exon is entirely included or excluded.

○ A5SS (Alternative 5’ or 3’ splice site): Alternative use of 5’ or 3’ splice junctions.

○ MXE (Mutually Exclusive Exon): One exon is included while the other is spliced out (common in brain tissue).

○ RI (Retained Intron): Non-coding intron is retained or spliced out.

○ Note: Variations in hemoglobin types are not due to alternative splicing but stage-specific expression.

⑥ 6th. Exon shuffling

○ Crossing over between different genes creates genes with new exon combinations.

⑦ 7th. Trans-splicing

Figure 12. cis/trans splicing

○ Type 1. Cis-splicing: Exons within a single pre-mRNA are spliced.

○ Type 2. Trans-splicing: Exons from different pre-mRNAs form a single mRNA molecule.

○ 2-1. SL trans-splicing: Spliced leader (SL) from 5’ tss joins 3’ tss of pre-mRNA.

○ 2-2. Trans-splicing between different genes or within the same gene.

○ SL trans-splicing process:

○ Step 1. SL RNA at 5’ tss binds to pre-mRNA at 3’ tss.

○ Step 2. Mature mRNA contains an SL exon with a TMG cap, translated in the cytoplasm.

○ Step 3. Y-shaped product (SL intron + pre-mRNA outron) forms, similar to lariat intron in cis-splicing.

○ Step 4. Y-shaped product rapidly degrades.

Figure 13. SL trans-splicing process

⑧ 8th. mRNA stability regulation (lifespan regulation)

○ Longer mRNA lifespan enables more protein translation.

○ Prokaryotic mRNA: ~2–3 minutes lifespan.

○ Eukaryotic mRNA: Lifespan of days to weeks.

○ Example 1. Hemoglobin mRNA: Remains intact while red blood cells live, accounting for 2/3 of total blood RNA.

○ Example 2. Transferrin (iron transport protein):

○ Iron response element (IRE) downstream of coding region (AT-rich).

○ Fe³⁺↑: IRE-BP inactivates → Poly A sequence removed → mRNA degrades.

○ Fe³⁺↓: IRE-BP activates → Poly A sequence preserved → mRNA stabilizes → Increased expression.

⑺ 5^th. rRNA, tRNA transcription

① rRNA is transcribed in phosphorus by RNA pol I

② mRNA, microRNA are transcribed in the nucleus by RNA pol II

③ tRNA is transcribed in the nucleus by RNA pol III

④ rRNA promoters exist far upstream of the gene

⑤ mRNA, promoter of microRNA exists near gene upstream

⑥ Promoters of tRNA exist near the downstream genes

⑦ Requires all TATA-binding protein

⑻ 6^th. rRNA processing, tRNA processing

① rRNA processing

○ Type 1. 45S rDNA → 18S rRNA + 5 in phosphorus.8S rRNA + 28S rRNA

○ Type 2. Cleaving unmethylated bases

② tRNA processing: slight cleavage of 3 ‘and 5’ from pre-tRNA

⑼ Regulation of Prokaryotic Transcription

① Open Reading Frame (ORF): Start code (ATG) and end code (3), 100 amino acids

○ Frames divided into three bases that can be translated into amino acid sequences

② Expression control elements

○ promoter (P), effector gene (O) (inhibitor site) consensus sequence

○ Activator spot ex. E. coli cAMP-CAP (Catabolite Activator Protein) complex recognition site

③ Operon (See. ⑼)

④ Regulation by Transcription Inhibitors

○ Inhibitors bind RNA promoters when they bind to an effector gene

○ Inductive operon: In the presence of an inducer, the inhibitor is bound to the working gene, allowing RNA polymerase to bind to the promoter to initiate transcription.

○ Example. Decomposition operon: Lactose operon (3 genes)

○ Inhibitory operon: In the absence of an inducer, the inhibitor is bound to the working gene, allowing RNA polymerase to bind to the promoter to initiate transcription

○ Example. Synthetic operon: Tryptophan operon

⑤ Regulation by Transcription Activators

⑽ Operon: Having ORFs for several structural genes under one promoter / operating region and being expressed simultaneously

① Summary

○ Inductive operon: The substance of interest finally promotes the expression of the operon

○ Inhibitory operon: The substance of interest finally inhibits the expression of the operon

○ Positive regulation: Modulation on activators, inducers of activators are activated by the substance of interest and inhibitory operons are inhibited

○ Negative control: Regulation of inhibitors, inducers of inducer-induced operons are inhibited by the substance of interest and inhibitory operons are activated

② Lactose operon (lac operon): Inductive operon

○ composition of lac operon: (regulator)-CAP binding site-promoter-working site-lac Z-lac Y-lac A

Figure 14. Lactose Operon Gene Composition

○ Regulator: Sites that encode inhibitors, because they have their own promoter, are not included in the operon (always expressed)

○ Operating site

○ Structural Gene 1. Beta galactosidase (lac Z)

○ Beta galactosidase: Lactate lyase converts lactose into allo lactose

○ Virtually the α peptide of the N-terminal part of beta galactosidase

○ α complementation: α peptide and other peptides in the host to form beta galactosidase

○ Structural Gene 2. Beta galactosid permease (lac Y): Lactose transporter, expressed on cell membrane

○ Structural Gene 3. Beta-galactoside acetyl group transferase (lac A)

○ Beta-galactoside acetyl group transferase: By-product removal

○ negative regulation of lac operon

○ Inhibitors of the lac operon are made from the lac I gene and inhibit the promoter binding of RNA pol

○ When lac inhibitors bind to allolactose, a variant of lactose, they do not bind to the working site (lactose inducers)

○ The promoter of lac operon has a weak affinity with RNA pol, resulting in poor transcription even without inhibitors

○ Positive regulation of lac operon

○ Adenylcyclic enzyme (AC) makes cAMP from ATP and there is an allosteric site of glucose

○ Metabolic activator protein (CAP) binds to cAMP to form a complex

○ CAP-cAMP complex assists promoter binding of RNA pol

○ lac operon provides a mechanism of diauxic growth in medium with glucose and lactic acid

○ Glucose O, Lactose O: decrease cAMP → decrease positive control → produce lactic acid lyase ×

○ Glucose O, Lactose X: decrease cAMP → decrease positive control → produce lactic acid lyase ×

○ Glucose X, Lactose O: Allolactose ↓ → binding of the inhibitor to the working site ↑ → production of lactic acid lyase

○ Glucose X, Lactose X: Allolactose ↓ → binding of the inhibitor to the working site ↑ → lactic acid lyase production ×

○ Operon derived from lac operon

○ tac operon. ③)

○ lac I^-: Inhibitor defective and unable to engage working area. Always active

○ lac I^S: Lactose did not bind to the inhibitor. Always inactive

○ lac O^c: Defective operating part prevents the suppressor from engaging the operating area. Always active

○ lac Z^-: Encrypt the defective lac Z. Β-galactosidase expression independent of operon ×

③ Tryptophan operon (trp operon): Inhibitory operon

○ composition of trp operon: (regulator)-promoter-operating part-structural gene

Figure 15. Tryptophan operon gene composition</center>

○ Regulator: Encoding the Suppressor. Not included in operon because it has its own promoter (always expressed)

○ Five structural genes encoding the five enzymes necessary for the biosynthesis process of tryptophan trp E, D, C, B, A

○ ξ polypeptide, δ polypeptide, indole glycerolphosphate synthase, β polar peptide, α polar peptide, respectively

○ Adjusting mechanism 1. Negative control

○ trp promoter requires relatively high binding affinity for RNA polymerase

○ The trp inhibitor binds to trp by the regulatory gene (trp R) and attaches to the working site (corepressor action).

○ Adjusting mechanism 2. Attenuation Control Mechanism

○ trp mRNA has four parts that can complementarily form base pairs. Each trp encryption site is nearby

○ Leading Sequence: Translated to protein but not related to the biosynthesis of tryptophan

○ In the term “leading sequence,” the meaning of “leading” implies being synthesized before the actual tripeptide enzyme.

Figure 16. DNA sequence of the trp operon

Figure 17. Leader peptide mRNA sequence of the trp operon

Following the poly-U sequence, trp E, D, C, B, A sequences are present.

○ Case 1. Tryptophan is low: Even if RNA pol is transcribed up to 4 times, tryptophan tRNA is insufficient, resulting in slow translation of the trp genetic code. Far away from transcription. No implicit terminator (above)

○ Case 2. Tryptophan rich: When RNA pol is transcribed up to 4 times, the translation speed is fast, and ribosomes are located at 1-2 sequences → 3 and 4 hairpin formation → Transcription termination

○ Case 3. If there is no tryptophan: Even if RNA pol is transcribed up to 4 times, no ribosome bound to mRNA is found → 3 and 4 hairpin formation → Termination of transcription

Figure 18. Hairpin Formation in the Attenuation Regulator of trp Operon

④ tac operon

○ Isopropylthiogalactoside (IPTG): Artificial substances that activate lac and tac promoters, such as allolactose. Maintain constant concentration and do not decompose, always activate promoter

○ Artificial promoter that requires IPTG like lac promoter

○ Stronger expression than trp promoter

○ Inhibitors are made from lac I or lac I^Q

○ lac I^Q: Inhibitor genes mutated to be stronger than lac I

⑤ PL operon

○ cI repressor (cI 857) gene: Inactivate PL promoter by making cI repressor at low temperature

⑥ T7 Operon (future update)

⑦ Diploid: cis-trans diploid

○ Diploid: If only part of the E. coli gene has an F plasmid, the genes in the F plasmid are diploid.

○ Partial dimer: Splicing. Partially considered to be embryo

○ cis-acting element: Promoters affecting only magnetic operons in partial diploids. Always same action

○ Example: Oc from lac-derived operon

○ trans-acting element: Promoters that affect both diploids and diploids. Conditional action

○ Example: I-, Is from lac-derived operon

⑧ Recombinant Protein and Inducible Promoter

○ Cells that continue to make recombinant proteins are metabolic: Cell growth inhibition, plasmid instability, decreased production yield

○ Expression strategy: After raising the cell concentration to some extent, inducing substance is added to induce expression

○ Conclusion: Inducible promoters that can control expression are widely used to increase production yield

⑾ Regulation of Eukaryotic Transcription

① Transcription factor

○ Eukaryotic RNA polymerase cannot bind to promoter alone

○ Various regulatory sequences upstream of the gene (eg: RNA polymerase starts transcription when universal transcription factor and special transcription factor bind to TATA box, CAAT box)

○ Universal Transcription Factor: Universally present in each cell (eg: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, TFIIJ, etc.)

○ Special Transcription Factor: Specifically present (eg: DNA-binding domain (DBD), activation domain (AD)

○ Short-range control element: Promoters are near

○ 1st. TFIID binds to the TATA box through the TBP subunit of TFIID.

○ 2nd. TFIIA and TFIIB stabilize the entire complex.

○ 3rd. TFIIF and RNAPII (RNA polymerase II) bind to the complex.

○ GTF is involved in recruiting RNAPII.

○ 4th. TFIIE and TFIIH additionally bind to the complex.

○ TFIIH phosphorylates the CTD (C-terminal domain) of RNAPII in the nucleus → Releases TFIID and activates helicase activity.

○ RNAPII, unlike α2ββ’ω, does not have helicase activity.

○ 5th. Transcription is initiated: PIC (pre-initiation complex) is formed.

○ GTF is also involved in transcription initiation.

Figure 19. PIC formation process

○ Remote regulator: mainly distributed upstream of the promoter

○ Enhancer (enhancer): Promote promoter binding of RNA polymerase

○ Silencer (silencer): RNA polymerase unconditional inhibition

○ insulator (insulator): Promotes transcription by suppressing enhancers

○ Stress reaction element (SRE)

○ DNA binding protein

○ HMG Protein: Bends the DNA so that DNA pol II can function

○ DNA binding motif

○ helix-turn-helix motif: Two alpha helices, CAP and operon operating sites

○ zinc finger motif: Observed in steroid hormones

○ leucine zipper motif (example: AP-1 is involved in mammalian cell growth and division)

○ helix-loop-helix motif

② Regulation of the Formulation Process (pre mRNA → mature mRNA)

③ DNA Methylation

○ Methyl group is attached to C base.

○ MBD1 ~ MBD6

○ MeCP2: Related to Rett syndrome.

○ 70-80% of the human genome is methylated.

○ global methylation map

Figure 20. Global methylation map

○ Chromatin with methyl groups attached condenses and becomes transcriptionally inactive.

○ Expression level: Euchromatin ＞ Heterochromatin.

○ Promoters and transcription factor binding sites have a high content of cytosine bases (e.g., GC box).

Figure 21. DNA methylation and transcription inactivation

④ Histone modification

○ Merging of histone with DNA

○ Among histones, H3 is the one where the most modifications can occur.

○ Histone acetylation

○ Positively charged amino acids lysine and arginine play a key role in histone condensation

○ NH₂ groups in the functional groups of lysine and arginine act as bases

○ Histone neutralization occurs when acetyl group (-COCH3) is bound to NH2 group of lysine and arginine

○ Histone neutralization → weakening electrostatic binding between DNA and histones → sedation and transcription activation

○ Histone deacetylation: Opposition of histone acetylation

○ Valproic acid: an inhibitor of histone deacetylase.

○ Histone methylation: Increased histone positive charge → DNA-histone electrical bond ↑ → Heterochromatinization and transcription inactivation

○ Histone phosphorylation: Different transcription activity and inactivation for each phosphorylation group

○ Histone Modifications and their Effects on Activation/Inhibition

○ Only lysine amino acids can undergo mono-, di-, tri-methylation.

○ As a result, the regulation of transcriptional activity is not straightforward.

Table. 2. Effects of Histone Modifications on Activation/Inhibition

For example, H3K27 refers to the 27th lysine amino acid in the H3 histone.

⑤ chromatin remodeling protein

○ remodeling protein a: Enable ATP

○ remodeling protein b: ATP not used

⑥ X chromosome inactivation

⑦ RNA interference (RNAi)

○ miRNA (micro RNA, micro RNA): If start is single stranded

○ 1^st. gene → pri-miRNA, RNA pol Ⅱ

○ 2^nd. pri-miRNA → pre-miRNA, Drosha, Pasha involved, pre-miRNA hairpin structure

○ 3^rd. pre-miRNA released extranuclear, RNA-GTP, Exportin-5 involved

○ 4^th. pre-miRNA → miRNA / miRNA * duplex, Dicer is involved, miRNA / miRNA is dsRNA

○ miRNA / miRNA * duplex is about 20 bp

○ 5^th. miRNA / miRNA * duplex → miRNA, Ago, RISC are involved, miRNA is ssRNA

○ RNA-induced silencing complex (RISC)

○ 6^th. miRNA binds to mRNA

○ miRNA and mRNA are completely complementary: mRNA degradation

○ miRNA and mRNA are partially complementary: stop mRNA translation

○ About 250 miRNAs present in humans

○ siRNA (small interfering RNA): Almost similar to miRNA but with double strand start

○ SiRNA: Refers to externally introduced pre-miRNA (shRNA) or dsRNA

○ 1^st. When introduced in the form of pre-miRNA: pre-miRNA → miRNA / miRNA * duplex, Dicer involved

○ 2^nd. miRNA / miRNA * duplex → miRNA, Ago, RISC are involved

○ 3^rd. miRNA binds to mRNA

○ Viral RNA or introduction RNA for experimental purposes

○ Any gene can suppress expression

○ Compare: miRNA has hairpin structure and siRNA has no hairpin structure

○ Animals have RNAi, while plants have PTGS, but not in prokaryotes and archaea

5. Translation

⑴ tRNA

① tRNA: 20 types of anticodons bind to mRNA codons

② Aminoacyl tRNA transpeptidase: 20 types

○ A specific amino acid is bound to the corresponding tRNA to produce an active amino acid (aa-tRNA)

○ Amino acid binds to the 3 ′ end of tRNA (using ATP): Dehydration Condensation of -COOH with 3’-OH Group

○ -COO- → -CO (AMP) → -CO (tRNA) (enzyme: Aminoacyl tRNA transpeptidase)

○ Amino acid binds to 3 ‘terminal pentose

○ If the amino acid and anticodone do not match, correction is possible, but not later

○ Only L-type amino acids are used. See, glucose in vivo is only D-type glucose.

Figure 22. Structure of tRNA

③ Primary transcripts of tRNAs become mature tRNAs through post-transcriptional modification

○ Mature tRNA: Single stranded RNA consisting of about 80 to 90 nucleotides

○ Some of the bases in the tRNA’s specific site are chemically modified (methylguanin, tibothymidine, dihydrouridine, etc.) and these modifications give stability.

○ Add three nucleotides (-CCA) to the 3 ‘end of tRNA

○ RNase D, a type of exonuclease

④ Mature tRNA has three rings

○ Because hydrogen bonds occur between the base and the OH group of ribose

○ Two-dimensional shape is planar clover leaf shape

○ Three-dimensional shape is L-shaped

○ 5 ‘end is shorter (because amino acid binds to 3’-OH group of tRNA)

⑤ Recognition Specificity of tRNA by Protein Synthesis Mechanism

⑵ rRNA

① Eukaryotes: Four different rRNAs are needed

○ 45S rDNA in phosphorus 45S rRNA (by RNA pol I) → 18S rRNA + 5.8S rRNA + 28S rRNA

○ 45S rDNA → 5S rRNA in phosphorous (by RNA pol III)

② Prokaryotes: Requires three rRNAs (5S rRNA, 16S rRNA, 23S rRNA), one RNA pol

③ Ribosome Assembly

○ Proteins synthesized from ribosomes in the cytoplasm are delivered to the phosphorus through the nuclear pores

○ Protein and rRNA assemble in phosphorus → two ribosomal subunits

○ Ribosome small subunit and large subunit are assembled after migration to cytoplasm (translation initiation complex)

④ Eukaryotic Ribosomes: 80S

○ 60S large subunit: 28S, 5.8S, 5S rRNA + 40 ~ 50 different proteins, tRNA binding site

○ A site (amino site)

○ P site (peptide site)

○ E site (exit site)

○ 40S small subunit: 18S rRNA + 30 protein, mRNA binding sites

⑤ Prokaryotes ribosomes: Contains about 55 proteins, 70S (high bacteria 70S ribosomes)

○ 50S large subunit: 5S, 23S rRNA

○ 30S small subunit: 16S rRNA

⑥ snoRNA is involved in rRNA processing

○ Bacteria: rRNA processing via rRNA cleavage, RNase III, RNase P, RNase E involvement

⑶ 1^st. Initiation: Ribosomes bind to the ribosome binding site (RBS)

① Initiation of prokaryotes

○ SD-Shine-Dalgarno sequence: Purine-rich sequence of 5’-AGGAGGU-3 ‘

○ 30S subunit (16S rRNA + protein) binds to Shine-Dalgarno sequence and moves until AUG of mRNA

○ Initiation tRNA (formyl-Met-tRNA) recognizes initiation codon of mRNA and binds to P site (using GTP)

○ Combination of Initiators (IF-1, IF-2, IF-3) with Large Monomers to Form Translation Initiation Complexes

② Initiation of eukaryotes

○ Begin with the subunit binding to the 5’-cap end of mRNA: 18S rRNA bound

○ Presence of consensus sequence called Kozac sequence before initiation codon

○ Met-tRNA binds to P-site after recognition of mRNA initiation codon (using GTP)

○ Initiators and large monomers combine to form translation initiation complexes

⑷ 2^nd. Elongation

① A site, P site, E site

○ A site: Site where aa-tRNA enters translation initiation complex

○ P site: Sites where peptide bonds are formed to lengthen amino acid chains

○ E site: tRNA exits the translation initiation complex

○ 5 ‘-E-P-A-3’ in order, don’t confuse

② Elongation factor: EF-Tu, EF-Ts, EF-G

③ codon recognition: Complementary tRNAs involve codon recognition of the A site, require 2 GTP, and involve EF-Tu and EF-Ts

④ peptide bond formation: Peptide bond formation with the carboxyl group of the peptide terminal of P site and the amino group of A site amino acid

○ According to peptide transferase activity of large monomer) (Amino acid synthesis direction: N → C

⑤ As a result, all of the amino acid chains of the P site are suspended in the tRNA of the A site.

⑥ Translocation: Ribosome moves to next codon (P → E release, A → P), 1 GTP required, EF-G involved

⑸ 3^rd. Termination

① Termination codons (UAA, UAG, UGA) → release factor binds to P site in the kidney process

② Releasing Factor (RF): Hydrolysis, recycling the binding between the tRNA that was in place and the last amino acid of the polypeptide

○ RF-1: UAA and UAG Recognition

○ RF-2: UAA and UGA Recognition

⑹ Genetic code

① Do not use duplicate translations: One codon translated only once

② Password Directionality: Codon reading in 5 ‘to 3’ direction based on mRNA

③ Initiation codon: Beginning of translation always begins at AUG (Met) of mRNA

④ nonsense codon: Of the 64 codons, 3 of the codons are termination codons (UAA, UAG, UGA), so the amino acid designation ×

⑤ Wobble hypothesis: There are 61 tRNAs in theory but about 45 in reality.

○ Associated with the deaminoation process where the A base turns into inosine acid (I)

○ Present when a particular codon forms a bond with a non-complementary tRNA

○ The sequence of the third codon is called the wobble sequence, and the binding with tRNA is called wobble pairing.

○ Theory: The first base of the anticodon and the third base of the codon do not bind strictly

⑥ Degeneration: As approximately 20 amino acids correspond to two or more codons (45), tRNA → amino acid

○ Codon usage: Even if the same amino acid is produced, the translation speed varies depending on the tRNA

○ Some studies identify the amount of tRNA in the pool and replace it with an appropriate codon to speed up translation

⑦ Unambiguity: By ⑤,⑥, one codon corresponds to one amino acid, mRNA → amino acid

○ Exception: One codon corresponds to multiple anticodons

⑧ universal codon

○ Genetic code evolved early in life’s history

○ Keeps unchanged because even minimal changes can be fatal

○ Some protozoa, found exceptions in mitochondria

⑨ overlapping gene: Viruses have a small amount of genomes, so information overlap (translational violations) is observed, one base interpreted up to six times, and one mutation has a significant effect

⑺ Genetic Code Decoding: 1968 Physiology Award

① amino acid: 20 kinds

○ ₄∏₂ = 16 < 20 < ₄∏₃ = 64

○ Early scientists deduce the unit of code is three nucleotides

② Type of genetic code

○ A meaningless password: Three stop codons serve as a period for the amino acid sequence

○ Meaningful passwords: 20 amino acid designation

○ Initiation codon: Methionine (AUG)

③ Decoding Genetic Code

○ Method 1. Marshall W. Nirenberg and J. H. Mattaei Experiment (1961)

○ Artificial Synthetic RNA and Cell-Free Protein Synthesis System

○ Amino Acid Decoding for UUU, AAA, CCC, GGG

○ Method 2. H. Gobind Khorana Experiment

○ Additional Genetic Code Reading Using Synthetic RNA

○ Example: 5’-CACACACACAC-3 ‘, ATP: CTP = 1: Threonine with a mixture of 1: Histidine = 1: 1

○ Method 3. Marshal W. Nirenberg and Ochora Experiment

○ UTP and GTP 3: Shuffled into one

○ UUU codon probability is 0.753 → the detection rate tells you what amino acid matches

○ Not sure which two codons have similar probability correspond to which codon

○ Example: 5’-CACACACACAC-3 ‘, ATP: CTP = 5: Lysine 100, threonine 26, asparagine 24, glutamine 24, proli 7, histidine 6 using a mixture of 1

○ Method 4. tRNA hybridization + filtration: Accurate codon tabulation, actually method 4 used

④ Genetic code table

Table 3. Genetic code table

⑻ Reading Frame (RF)

① Definition: A frame separated by 3 nucleotides that can be translated into an amino acid sequence.

② Composition: Approximately 100 amino acids starting from the start codon (ATG; met).

③ Open Reading Frame (ORF): Multiple reading frames generated based on how certain codons are considered as start and stop codons.

④ Reading frames do not include stop codons (there are 3 types).

⑤ Position of reading frames within mRNA.

Figure 23. Position of reading frames within mRNA.

6. Delivery of Proteins After Translation

⑴ Overview

① Synthesized proteins contain signal sequences composed of specific amino acid sequences, directing the protein to specific locations within the cell.

② Field awarded the Nobel Prize in Physiology or Medicine in 2013.

⑵ Intracellular compartments

① Intracellular chemical reactions are often opposed to each other and require intracellular compartments

② How to assemble several enzymes that co-catalyze a specific reaction process into one large protein complex (prokaryotic, eukaryotic)

③ Partitioning in one membrane structure (eukaryotic): Significance of mesenchymal organelles

④ Transfer of newly synthesized protein to corresponding mesenchymal organelles (protein targeting)

⑶ Signal Sequences

① Typically around 20 sequences located at the N-terminus.

② Vesicle-targeting signal sequence: Approximately 10 hydrophobic amino acids.

○ Reason for hydrophobicity: Hydrophilic signal peptides have difficulty passing through membranes.

③ Nuclear localization signal sequence: Consists of consecutive (four or more) amino acids with a positive charge.

④ Chloroplast targeting signal sequence: Involves two different signal sequences.

⑤ Example: Signal sequence of Hsp90.

Figure 24. Signal sequence of Hsp90.

○ All Hsp90 proteins include a signal sequence composed of 10 amino acids.

○ Y (1), F (7), R (9) are conserved in all species.

○ The size of each amino acid symbol in the graph below is proportional to its frequency.

○ Theoretically, the second amino acid can be any amino acid, but Ser is the most commonly found.

⑷ Free ribosomes (free ribosom)

① Floating in the cytoplasm and synthesizing proteins acting inside the cell

② Characteristic

○ Typically Protein Delivery Mechanism ×

○ Proteins produced by free ribosomes are free of disulfide bonds

○ post-translation: Proteins move after translation is complete

③ Example 1. Nuclear protein

○ → inside and outside the protein through the nuclear pore: nuclear export signal + exportin

○ → out of protein through nuclear pores: nuclear localization signal + importin

○ Preservation of signal sequence and preservation of tertiary structure because receptor protein is transported together after binding to protein signal sequence

○ Nuclear pores move into a folded state when entering and leaving a protein

○ Use GTP when repositioning importin and exportin

④ Example 2. peroxisome protein: Preservation of tertiary structure and signal sequence by peroxine

⑤ Example 3. mitochondria and chloroplast proteins: Tertiary structure is broken when protein moves to membrane, cutting signal sequence

○ Proteins delivered to chloroplast have two signal sequences

⑥ Example 4. Superficial proteins

⑦ Example 5. Cytoplasmic protein: Cytoskeleton protein, etc.

⑸ Bound ribosom

① Signal sequence

○ Typically about 20 sequences present at the N-terminal site

○ Vesicular Signal Sequence: About 10 hydrophobic amino acids

○ Nuclear batch signal sequence: Amino acids with consecutive (+) charges

② Protein Delivery Mechanisms

○ 1^st. Start translation

○ 2^nd. co-translation: Signal peptide on the amino acid sequence being translated binds to SRP in the cytoplasm

○ 3^rd. Move after removing the line: Signal peptidase removes signal sequence

○ 4^th. Move after removing the line: Entry into the endoplasmic reticulum through a mobile complex

○ 5^th. Resume translation

③ Example: Membrane protein, exocrine protein, lysosomal protein

7. Post-translational Modification (PTM)

⑴ Overview

① Purpose 1: Regulation of Activity

○ Post-translational modifications (PTMs) can increase or decrease protein activity.

○ PTMs can confer various functions.

② Purpose 2: Protein-Protein Interaction

○ Sites modified by PTMs can be binding interfaces.

③ Purpose 3: Subcellular Interaction

○ PTMs can serve as targeting signals or membrane anchors.

④ Purpose 4: Lifespan Regulation

○ PTMs can promote protein degradation or scavenging.

⑵ Chemical Modifications

① Glycosylation: Most membrane and signaling proteins are glycoproteins.

○ Classification 1 : Differences in glycosylation processes by domain.

○ E. coli: No glycoproteins due to the absence of glycosylation processes.

○ Yeast: Glycoproteins exhibit a form with abundant mannose.

○ Yeast Structure: Cytosol - Cell membrane - Chitin - β-Glucan - Mannose.

○ Animal cells: Glycoproteins exhibit a form with sialic acid.

○ Classification 2 : Intracellular glycosylation processes - N-glycosylation and O-glycosylation.

○ N-glycosylation:

○ Mainly targets cell membrane proteins, attaching N-glycan.

○ Site: Asparagine (Asn) (N) binding in the endoplasmic reticulum.

○ Attaches multiple sugars.

○ Albumin does not undergo N-glycosylation.

○ PNGase F: Degrades N-glycosylation.

○ O-glycosylation:

○ Mainly targets nuclear and cytoplasmic proteins, attaching O-glycan.

○ Site: Serine (Ser), Threonine (Thr) (O) binding in the Golgi apparatus.

○ Attaches a single sugar.

○ Types: O-N-acetylgalactosamine (O-GalNAc), O-N-acetylglucosamine (O-GlcNAc), O-mannose (O-Man), O-galactose (O-Gal), O-fucose (O-Fuc), O-glucose (O-Glc).

○ Glycocapture Method: Determines the site of N-glycosylation.

② Phosphorylation: Crucial for signal transduction.

③ Sulfation: Occurs in the trans-Golgi network, involving protein disulfide isomerase (PDI).

④ Prenylation: Includes farnesylation, geranyl-geranylation, and others.

⑤ Ubiquitination: Refer to additional information.

⑥ Acetylation: Example: Lysine → Acetyl-lysine.

⑦ Other modifications: Methylation, nitration, amidation, formylation, palmitoylation, hydroxylation, SUMOylation, lipid anchoring.

⑶ Chaperones

① Translated protein precursors acquire the appropriate folding structure, forming tertiary and quaternary structures with the help of chaperones.

② Example 1 : Partial degradation

○ Example: Cleavage of Signal Sequences, Protein Activation by Endopeptidases, etc.

○ Example: Insulin - Amino acid cleavage is necessary for insulin activation.

○ In the rough endoplasmic reticulum, the N-signal of prepro-insulin is cleaved, forming pro-insulin.

○ In the Golgi apparatus, pro-insulin undergoes cleavage.

○ Insulin and C-peptide are secreted separately.

○ Insulin and C-peptide are clinically useful for diagnosis.

○ Example: Enteropeptidase Attached to the Intestinal Barrier.

○ Trimming some of the trypsinogen to transform it into trypsin.

○ Trypsin cuts some of the chymotrypsinogen to transform it into chymotrypsin.

③ Example 2 : Calnexin - Precise tertiary structure associated with cystic fibrosis.

④ Example 3 : Heat Shock Proteins (HSPs)

⑷ Zymogen

① Inactive enzyme precursors.

② Acquire activity through post-translational processing after translation.

③ Most digestive enzymes are in zymogen form.

⑸ Protein Synthesis Regulation

① Example 1: Ferritin - Iron storage protein.

○ Iron Response Element (IRE) is present in the encryption region upstream of the iron-responsive element-binding protein (IRE-BP).

○ Fe level ↑: IRE-BP is inactive → protein translation initiation → expression.

○ Fe level ↓: IRE-BP is active → IRE-BP binds to mRNA RBS → protein translation inhibition.

○ Dps: Functions as ferritin in prokaryotes, discovered in E. coli.

⑹ Lifespan Regulation

① Purpose

○ Proteins with incorrect or denatured structures may be toxic in the body.

○ Many proteins are degraded even if they are normal to regulate the activity of intracellular proteins.

② Example 1 : Ubiquitin - A small protein composed of 76 amino acids.

○ Ubiquitin attaches to the protein to be degraded and poly-peptide with chaperone (e.g., hsp70) assistance.

○ The protein tagged with ubiquitin is recognized by proteasome and then undergoes degradation.

③ Example 2 : Lysosome and Mannose Pathway - Digestion of targeted molecules, damaged receptors, and damaged organelles.

④ Example 3 : PROTAC (PROteolysis TAgeting Chimeras)

○ Overview

○ Definition: A technology that utilizes the Ubiquitin-Proteasome System to degrade proteins, including those not previously targeted for inhibition.

○ ProTACs are molecules that artificially link a chemical compound to E3 ligase and to the protein targeted for degradation.

○ Molecular glue components forming ProTACs were accidentally discovered while searching for drugs with physiological activity.

○ E3 Ubiquitin Ligase

○ Attaches ubiquitin to the protein targeted for degradation.

○ Although there are around 600 types, only 2-3 are actively used as ProTACs.

○ Composition

○ Compound binding to E3 Ubiquitin Ligase.

○ Compound binding to the protein targeted for degradation.

○ Linker connecting the two compounds.

○ Examples

○ VHL (von Hippel-Lindau tumor suppressor): Widely used in ProTAC technology.

○ CRLs·Cullin-RING E3 ligase: Induces degradation by attaching ubiquitin to various proteins.

○ SD-36: A ProTAC selectively degrading STAT3, a transcription factor involved in the growth, proliferation, invasion, and metastasis of cancer cells.

○ Fulvestrant: Induces degradation of estrogen receptor (ER) by inhibiting ER and attaching a degradation-inducing moiety.

○ Thalidomide: A representative example of molecular glue.

○ Lenalidomide, Pomalidomide: Derivatives of thalidomide functioning as both thalidomide inducers and molecular glue.

8. Inhibitor

⑴ DNA Polymerase Inhibitors

① Acyclovir (acycloguanosine): Antiviral drug that inhibits viral DNA polymerase (tk) reaction, hindering viral DNA synthesis.

② AraC (cytosine arabinoside, cytarabine): Anticancer drug.

③ AraA (adenine arabinoside, vidarabine): Antiviral drug.

④ Antinomycin D: Binds to the groove, preventing DNA replication; can act on both prokaryotic and eukaryotic cells.

⑤ Fluoroquinolones (ciprofloxacin): Inhibits DNA gyrase.

⑥ Carboplatin: Anticancer drug that inhibits DNA synthesis.

⑦ Topoisomerase I Inhibitors: Inhibitors of type IB topoisomerase.

○ Irinotecan

○ Topotecan

○ Camptothecin (CPT-11: Camtosar)

○ Diflomotecan

○ Lamellarin D

⑧ Topoisomerase II Inhibitors

○ Etoposide

○ Teniposide

○ Doxorubicin

○ Daunorubicin

○ Mitoxantrone

○ Amsacrine

○ Ellipticines

○ Aurintricarboxylic acid

○ HU-331

○ Ofloxacin (Trade name: Tarivid)

○ Levofloxacin (Trade name: Cravit)

⑵ Transcription Inhibitors

① Antinomycin D: Transcription inhibitor.

⑶ Translation Inhibitors

① Cycloheximide: Translation inhibitor in eukaryotic cells.

② Translation Inhibitors Acting on Bacteria: Can also function as antibiotics.

○ Chloromycetin: Inhibits peptide bond formation.

○ Erythromycin: Inhibits ribosomal movement on mRNA.

○ Neomycin: Interferes with tRNA-mRNA interaction.

○ Streptomycin: Inhibits translation initiation.

○ Tetracycline: Inhibits tRNA binding to the ribosome.

⑷ Nucleoside Reverse Transcriptase Inhibitors (NRTI)

① AZT (Zidovudine)

② Combivir (AZT + Epivir)

③ Emtriva (Emtricitabine)

④ Epivir (Lamivudine)

⑤ Epzicom (Abacavir + Epivir)

⑥ Hivid (ddC)

⑦ Trizivir (Abacavir + AZT + Epivir)

⑧ Videx & Videx EC (ddI): 2’, 3’-dideoxynosine, removes 3’-OH.

⑨ Zerit (D4T)

⑩ Ziagen (Abacavir)

Input: 2015.7.02 17:56