Korean, Edit

Chapter 8. Central Dogma

Higher category: 【Biology】 Biology Index 

1. Overview

2. Genes and Gene Expression

3. DNA replication

4. Transcription

5. Translation

6. Delivery of Proteins After Translation

7. Post-translational Modification

8. Inhibitor

a. DNA proofreading and repair

b. Central dogma of microbiology

1. Overview 

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

⑵ After the discovery of reverse transcriptase in retroviruses (RNA viruses), the central dogma was revised.

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 One gene, multiple polypeptides theory

① One gene, one enzyme hypothesis: Beadle and Tatum’s experiment

○ Inferring biosynthetic pathways using mutants: ornithine, citrulline, arginine

Table 1. Beads and Tatum’s Experiments

○ Tip 1. The more growing the material, the later stages of the biosynthetic pathway.

○ Tip 2. The more a mutant grows, the earlier the step it is involved in the metabolic pathway.

② One gene, one protein hypothesis: Proteins that do not function as enzymes are found.

③ One gene, one polypeptide hypothesis: Proteins forming quaternary structures are revealed.

④ One gene, multiple polypeptides hypothesis: It has been revealed that multiple polypeptides can be produced from a single 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 transcribes only specific genes, resulting in different gene expression patterns, which lead to variations in structure and function.

② Regulation of Transcription**

○ Regulation through transcription repression (prokaryotic cells)

○ Regulation through transcription activation (prokaryotic and eukaryotic cells)

○ Regulation through chromatin condensation: preventing RNA polymerase access (Barr bodies)

③ Regulation through mRNA degradation

④ Regulation of Translation

⑤ Regulation through protein degradation (proteases)

3. DNA replication

⑴ 1^st. Untangling 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 fork: The junction between the strand where the DNA is hydrogen-bonded and the strand that is not.

○ Prokaryotes: DnaA recognizes one replication origin.

○ DnaA

○ Consensus sequence: 9 base pairs × 4 + 13 base pairs × 3

○ 20-50 monomers

○ Binds simultaneously to multiple AT-rich sites.

○ Melts DNA by using one molecule of ATP.

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

○ The replication speed is slow, so there are multiple origins of replication.

○ Yeast: 250 to 400 replication origins.

○ Mammals: 25,000 origins of replication; approximately 6,500 in humans.

○ Rich in A-T sequences.

○ The unit of replication determined by the origin of replication is called a replicon.

○ ARS (autonomic replicatoin sequence)

○ DnaA and ORC create replication bubbles, exposing several dozen nucleotides of single-stranded DNA (ssDNA).

② Enzyme: Helicase, DNA topoisomerase, single chain binding protein

○ Helicase: Releases hydrogen bonds in two complementary strands.

○ Prokaryotes: DnaB, PcrA

○ Eukaryotes: MCM (Minichromosome Maintenance Complex)

○ DnaB

○ Helicase characteristic experiment: Two conclusions can be drawn

○ Conclusion 1: Requires replication bubbles created by DnaA or ORC to bind to DNA.

○ Conclusion 2: Typically binds to the template of the lagging strand at the replication fork and moves in the 5’ → 3’ direction (exceptions exist).

○ ATP is used.

○ Minimum ssDNA length for a replication bubble: 16 nucleotides or more.

○ 100 turns/second = 6,000 rpm

○ PcrA: Consists of A1, A2, B1, B2 domains, and a P-loop.

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

Figure 1. Topological isomers of circular DNA with a size of 210 bp

The second double helix has fewer nucleotides per turn (lower torsional stress), while the third double helix has more nucleotides per turn (higher torsional stress).

○ L (Linking number): The number of loops. Conserved during torsional stress calculations, with counterclockwise (+) rotation.

○ T (Twist number): The number of helical turns. DNA is inherently right-handed, so it has counterclockwise (+) torsional stress.

○ W (Writhe number): The number of supercoils.

○ Supercoil: A structure where the DNA molecule forms loops on itself, related to the enhancement or relaxation of torsional stress.

○ Positive supercoil: A left-handed supercoil (the third in the figure), with a positive writhe number.

○ Negative supercoil: A right-handed supercoil (the second in the figure), with a negative writhe number.

○ Positive supercoils form left-handed supercoils, thereby increasing the right-handed torsional stress of the double helix.

○ Negative supercoils form right-handed supercoils, thereby relaxing the right-handed torsional stress of the double helix.

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

○ DNA topoisomerase: An enzyme that relieves torsional stress in DNA.

○ Acts at a region far ahead of the replication fork.

○ Type 1 DNA Topoisomerase

○ An enzyme that cuts one strand of the DNA, allows it to unwind naturally, and then reseals it.

○ Strand-cutting and strand-releasing activities: Nuclease and ligase activities.

○ Does not require ATP.

○ Relieves negative supercoils in E. coli.

○ Step 1. Tyrosine attacks the phosphate backbone, creating a nick in the DNA strand.

○ Step 2. The DNA rotates one turn under torsional stress.

○ Step 3. Resealing occurs, ultimately resolving the supercoil.

○ Type 2 DNA Toposiomerase (DNA gyrase)

○ An enzyme that cuts both strands of the DNA, rotates (topologically rearranges) them, and reseals them.

○ Converts positively supercoiled DNA molecules into negatively supercoiled forms.

○ Requires ATP hydrolysis.

○ Targeted by antibiotics such as fluoroquinolones (e.g., ciprofloxacin).

○ Single-Stranded Binding Protein

○ Prokaryotes: Single-Strand Binding Protein (SSBP); a monomer with two domains.

○ Eukaryotes: Replication Protein A (RPA).

○ Function 1. Temporarily binds to each single strand of DNA after the hydrogen bonds are broken, preventing the strands from pairing back together.

○ Function 2. Stabilizes the DNA.

○ Function 3. Protects DNA from twisting.

○ SSBPs are required to activate the helicase function in experimental settings.

③ Prokaryotic cells: Circular DNA, one replication origin.

④ Eukaryotic cells: Linear DNA, multiple replication origins.

○ Eukaryotic cells have larger genomes than prokaryotic cells, so they have many origins of replication.

⑵ 2^nd. Attaching primer

① Primer: Short RNA fragments underlying DNA polymerization

○ PCR primers are DNA fragments.

○ The primer is approximately 10 nucleotides in length.

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

③ Enzyme: Primase is involved. Primase is responsible for synthesizing and attaching primers.

○ Since helicase operates at a speed of 100 turns/second, primase also works at a remarkably fast pace.

○ Eukaryotes: Pol α/primase complex.

④ Primosome: A prepriming complex formed by several proteins before the synthesis of RNA primers.

⑶ 3^rd. DNA polymerization

① Enzyme: DNA polymerase is involved.

○ E. coli has three DNA polymerases: 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: Repairs DNA damaged by external factors and is activated when DNA synthesis is interrupted.

○ DNA pol III: Involved in DNA elongation. Functions as a dimer and does not move itself; instead, the DNA strand moves.

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

○ 1000 nt/s

○ Asymmetry: Proceeds differently when elongating the lagging strand and the leading strand.

○ 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: Related to sliding DNA clamp.

○ Sliding clamp

Figure 2. Sliding clamp

○ Ensures proper alignment of the template strand and the daughter strand, allowing DNA polymerase to synthesize DNA accurately and efficiently, enhancing both precision and efficiency.

○ Eukaryotes: The sliding clamp is associated with PCNA (Proliferating Cell Nuclear Antigen), which is related to Ki-67 staining.

○ Prokaryotes: The sliding clamp is temporarily opened by the clamp loader to allow the insertion of the closed circular structure of E. coli DNA into the central channel. It then closes again, enabling polymerase activity during DNA replication.

○ Klenow fragment

○ Recall DNA pol I: 5’ → 3’ polymerization, 3’ → 5’ exonuclease, 5’ → 3’ exonuclease.

○ Recall the large fragment of DNA pol I: 5’ → 3’ polymerization, 3’ → 5’ exonuclease.

○ Recall the small fragment of DNA pol I: 5’ → 3’ exonuclease.

○ Recall 5’ → 3’ exonuclease: Removes terminal nucleic acids.

○ Recall 3’ → 5’ exonuclease: Proofreading function.

○ The Klenow fragment is the large fragment of DNA pol I and is identical to DNA pol III.

○ The Klenow fragment is DNA polymerase of T4 phage.

○ Converts sticky ends of restriction fragments to blunt ends in DNA recombination.

○ Lacks 5’ → 3’ exonuclease activity, so it cannot degrade the primer of template DNA.

○ Example of an in vitro polymerization reaction using Klenow fragment.

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

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

○ DNA pol α: Contains primase, and lacks proofreading.

○ DNA pol β: Repair function, and lacks proofreading.

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

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

○ DNA pol γ: Replicates mitochondrial DNA, and has 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.

○ Pyrophosphate

④ A pairs with T, and G pairs with C in a complementary manner (purine-pyrimidine pairing → the distance between the two strands of DNA remains constant).

⑤ Composite direction: 5’ → 3’

⑥ Leading strand

○ A strand synthesized continuously.

○ Occurs when the direction of replication is the same as the movement of the replication fork.

⑦ Lagging strand

○ A strand synthesized in discontinuous fragments (Okazaki fragments).

○ Occurs when the direction of replication is opposite to the movement of the replication fork.

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 primer removal enzymes

○ Prokaryotes: DNA polymerase I

○ Eukaryotes: RNAase H, FEN1 (flap endonuclease)

② After RNA primers are removed, DNA ligase (DnaG) performs the DNA strand-joining reaction.

○ Ligase uses ATP.

○ Collectively, DnaB and DnaG are referred to as the primosome.

○ Step 1. E + ATP (or NAD⁺) → E-AMP + PPi (or NMN)

○ Activated AMP from ATP forms a phosphoamide bond with the e-amino group of a lysine residue in DNA ligase.

○ Step 2. E-AMP + ⓟ-5’-DNA → E + AMP-ⓟ-5’-DNA

○ Step 3. DNA-3’-OH + AMP-ⓟ-5’-DNA → DNA-3’-O-ⓟ-5’-DNA + AMP

○ The activated 5’ phosphorous atom undergoes a nucleophilic attack by the 3’-OH group.

③ Removal rate

○ 1 per second

○ 1 error per 10,000,000

④ The RNA primer at the 5’ end of the daughter strand is not replicated.

○ This is why DNA becomes shorter with successive rounds of replication.

○ Related to aging.

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

① Original marker: Marks the original template strand by methylating the A bases.

○ Dna methylase is involved. Methylation of 5’-GATC-3’ sequence at the ori C site.

② Proofreading: DNA polymerase has proofreading functionality.

○ Acts like replacing a single nucleotide.

○ 3’ → 5’ exonuclease activity

○ Performed by DNA pol I, II, and III.

○ Reduces replication error rate to 1/10⁷ during polymerization.

○ Step 1. During the DNA synthesis process, an incorrect nucleotide is incorporated, leaving the 3’ end of that nucleotide exposed at this stage.

○ Step 2. Structural fluctuation occurs significantly.

○ Step 3. Weakened hydrogen bonds allow the incorrect nucleotide to move to the exonuclease site.

○ Step 4. The incorrect nucleotide is removed through hydrolysis.

○ 5’ → 3’ exonuclease activity

○ Performed only by DNA pol I.

○ It replaces single nucleotides one at a time, but ultimately replaces an RNA primer approximately 10 nucleotides long in a single process.

○ Step 1. The polymerase positions itself between the 3’ end of the Okazaki fragment and the 5’ end of the adjacent RNA primer.

○ Step 2. Proceeds nucleotide by nucleotide:

○ Step 2-1. Removes the RNA primer through hydrolysis.

○ Step 2-2. Replaces each RNA nucleotide with dNTP using 5’ → 3’ polymerase activity and performs proofreading using 3’ → 5’ exonuclease activity.

○ Step 3. Repeats these steps until the gap is completely filled with DNA.

○ The HIV virus lacks a repair mechanism.

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

○ Like replacing multiple nucleotides.

○ Type 1. Dimer repair.

○ Type 2. Excision repair: Removes abnormal bases caused by chemical damage.

④ If DNA proofreading and repair fail, mutations occur frequently.

⑺ Characteristics of DNA replication

① Conservative replication: After replication, one original DNA molecule and one entirely new DNA molecule are present (rejected).

② Dispersive replication: After replication, two completely new DNA molecules are generated (rejected).

③ Semi-conservative replication: After replication, each DNA molecule contains one template strand and one newly synthesized strand (accepted).

④ Complementarity: The template strand and the newly synthesized strand contain complementary information.

⑤ Meselson-Stahl experiment: Validated the semi-conservative model of DNA replication.

Figure 4. Meselson-Stahl’s experiment

○ Density gradient centrifugation using cesium chloride → Separates DNA based on density.

○ E. coli labeled with ¹⁵N was cultured in a medium containing normal nitrogen (¹⁴N), and the DNA density was analyzed at each generation.

⑻ Telomerase: 2009 Nobel Prize in Physiology or Medicine

① The RNA primer at the 5’ end of the daughter strand cannot be replicated → DNA shortens → Aging

○ Hayflick limit: The number of cell divisions until the cell can no longer divide.

② Telomere

○ Protects DNA to some extent from shortening.

○ Consists of meaningless repetitive sequences. 6-nt long repeats. Repeated approximately 300 to 5000 times.

○ Tetra-G on the telomere forms a t-loop, transforming the exposed 3’ end of the telomere into a hairpin structure to protect the ends.

○ Humans, mice, birds, red bread mold, silkworms: TTAGGG

○ Arabidopsis thaliana: TTTAGGG

○ Chlamydomonas: TTTTAGGG

○ Yeast: TTAC(A)(C)G(1-8)

③ Telomerase

○ A reverse transcriptase and RNA-dependent DNA polymerase.

○ Extends telomeres using a short RNA template within the molecule.

○ The internal short RNA: 3’-CCCAAUCCC-5’ RNA template

Figure 5. Mechanism of exposed 3’ terminus in telomeres and telomerase

④ Found in germ cells, proliferating normal cells, and cancer cells.

⑤ In mice, telomerase is commonly found, unlike in humans.

⑥ Circular DNA (e.g., mitochondrial DNA) does not require telomerase.

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

Figure 6. rolling-circle replication

⑽ Multi-fork replication

① Situation: Under extremely favorable conditions, the reproduction cycle of E. coli is 20 minutes.

② Question: Since the DNA replication speed is 1000 nucleotides per second, it takes longer to replicate the entire chromosome than the reproduction cycle.

③ Solution: Multi-fork replication. This phenomenon allows replication to start on already replicating daughter strands, preparing for the next cell cycle before the current cycle is completed.

4. Transcription

Figure 7. Gene transcription

⑴ DNA vs. RNA

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

② Sugar: DNA contains deoxyribose (a 5-carbon sugar), while RNA contains ribose (a 5-carbon sugar).

○ Deoxyribose has a -H group on the 2nd carbon, while ribose has a -OH group on the 2nd carbon.

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)

○ tRNA (transfer RNA)

○ rRNA (ribosome RNA)

○ SRP RNA

○ snRNA (small nuclear RNA)

○ 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

○ piRNA

○ eRNA

○ lincRNA

⑵ Prokaryotic and Eukaryotic mRNAs

① Polycistronic and Monocistronic

○ Prokaryotes: Polycistronic mRNA (= operon)

○ A single mRNA molecule encodes several different polypeptide chains.

○ Each polypeptide has a start site containing a ribosome binding site (RBS).

○ Advantage: Allows multiple genes to be regulated by a single promoter.

○ Polyribosome (polysome): A structure where multiple ribosomes are bound to a single mRNA.

○ Eukaryotes: Monocistronic mRNA

○ Polyribosomes are observed but are extremely rare.

○ rRNA: Polyribosomes are also observed in eukaryotic cells for rRNA.

② Presence of introns: Found only in eukaryotes.

○ Histone DNA: Lacks introns.

③ Simultaneity of transcription and translation: Occurs only in prokaryotes.

④ Post-transcriptional modification: Present only in eukaryotes.

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 can encode genes, but any given gene uses only one strand as the template.

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

○ Sense strand (coding strand, (+) strand, non-template strand): The strand to which RNA polymerase does not bind, making it convenient for codon analysis.

○ Anti-sense strands (non-coding strands, (-) strands, template strands): The strand to which RNA polymerase binds.

○ 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.

○ Prokaryotic promoter consensus

○ Example 1. Pribnow box (-10 box): TATAAT (or TATGTT, ⋯)

○ Example 2. -35 box: TTGACA (or TTTACA, ⋯)

○ Eukaryotic promoter consensus

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

○ Example 2. CAAT box (-75 box): CAAT

○ Example 3. GC box

○ Core promoter: The region of approximately 300 bp from the promoter to just before the start of transcription.

④ Upstream / downstream

○ The first nucleotide transcribed into RNA is designated as +1.

○ Based on +1, the 5’ direction is referred to as upstream (-), and the 3’ direction as downstream (+).

⑤ Sigma factors in E. coli: All recognize the -35 upstream region.

○ σ⁷⁰ (σ^D): Recognizes TTGACA. The primary sigma factor involved in most genes during normal growth.

○ σ⁵⁴: Recognizes TTGGCACA. Involved in nitrogen anabolism.

○ σ³⁸: Recognizes CCGGCG. The major sigma factor involved during stationary phase, oxidative stress, and osmotic stress.

○ σ³²: Recognizes TNTCNCCTTGAA (N represents any nucleotide). Involved during heat shock stress.

○ σ²⁸: Recognizes TAAA. Involved in the synthesis of flagellar genes.

○ σ²⁴: Recognizes GAACTT. Involved in the response to misfolded proteins in the periplasm.

○ σ¹⁹: Recognizes AAGGAAAAT. Involved in genes responsible for iron ion transport.

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

① Transcription bubble = RNA transcript + DNA + RNA polymerase

② RNA-DNA hybrid

③ RNA polymerase: No primer required. No topoisomerase. No exonuclease activity leading to a higher transcription error rate.

④ E. coli has one type of DNA-dependent RNA polymerase.

○ Holoenzyme form: α2ββ’ωσ

○ Core enzyme form: α2ββ’ω. Binds to the promoter with the help of the σ⁷⁰ factor, which recognizes the promoter.

○ σ⁷⁰: Recognizes the promoter and initiates synthesis. Can be reused.

○ β: Forms phosphodiester bonds and binds to rNTPs.

○ β’: Binds to the DNA template.

○ Core enzyme: Only involves the α subunit.

○ No specific binding occurs.

○ Tight non-specific DNA binding.

○ K_d ≈ 5 × 10^-12 M

○ Holoenzyme: Includes the σ⁷⁰ subunit

○ Specific promoter binding occurs.

○ Weak non-specific DNA binding.

○ K_d ≈ 10^-7 M

○ Finds the promoter 10,000 times faster.

⑤ Eukaryotes have three types of DNA-dependent RNA polymerases.

○ RNA pol I: Functions in the nucleolus. Synthesizes 28S rRNA, 18S rRNA, and 5.8S rRNA.

○ RNA pol II: Functions in the nucleoplasm. Synthesizes mRNA.

○ RNA pol III: Functions in the nucleoplasm. Synthesizes tRNA, 5S rRNA, ncRNA, and snRNA.

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

① Prokaryotic Terminators

○ Intrinsic Terminator: Transcription is terminated by forming a hairpin (hairpin-oligo-U structure).

○ A hairpin is formed due to an inverted repeat sequence.

○ An A-T rich site is located near the inverted repeat sequence, where U bases are transcribed using A as the template.

○ The transcription product naturally dissociates due to the unstable A=U base pair chain.

○ Rho-dependent Terminator: The Rho factor acts as a DNA-RNA helicase to promote the detachment of the RNA being synthesized.

② Eukaryotic Terminators

○ When the poly-A signal sequence (5’-AAUAAA-3’) appears on the transcript, endonuclease activity is triggered.

○ The endonuclease cleaves the transcript 10 to 35 nucleotides downstream, leading to its detachment.

○ A poly-A tail, consisting of multiple A bases, is added to the cleaved 3’ end.

⑹ 4^th. mRNA processing (eukaryotic cells only): Mature mRNA moves to the cytoplasm through the nuclear pore. Due to the maturity, the degradation is decreased and the translation is increased.

① 1^st. Capping

Figure 8. 5’-cap

○ Capping begins simultaneously with transcription.

○ 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: 5’-cap is 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)

○ 1^st generation: mCap (Cap 0) derived from yeast. The disadvantage is the lack of directionality.

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

○ 3^rd generation: Trinucleotide cap analog (e.g., CleanCap) (Cap 1). It is excellent because it closely resembles the 5’-cap found in vivo.

② 2^nd. 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 3^rd. Splicing

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

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

○ Introns: Encode non-practical 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.

○ That is, 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: As a result, a guanosine molecule binds to the intron, and the 5’ exon obtains 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.

○ In this way, the exons gradually become connected.

○ 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.

○ In this way, the exons gradually become connected.

○ Difference 1. Initial nucleophile: Group I uses an external guanosine molecule, while Group II uses an internal adenosine molecule.

○ Difference 2. Structure: Group I is characterized by a complex loop and helix structure, while Group II is characterized by a lariat structure.

○ Difference 3. Distribution

○ Group I: rRNA, mRNA, and tRNA of bacteria; mitochondrial genome and chloroplast genome of lower eukaryotes; rRNA of the nuclear genome in lower eukaryotes; and some tRNA and mRNA in the chloroplast and mitochondria of higher plants.

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

○ 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: Altering the RNA sequence

○ 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).

○ In humans, apolipoprotein B protein becomes 100 kDa Apo-B100 in the liver.

○ In humans, apolipoprotein B protein becomes 48 kDa Apo-B48 in the small intestine.

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

⑤ 5^th. 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 appear as a result of splicing → Removal of abnormal mRNA by NMD (nonsense-mediated mRNA decay).

○ Alternative splicing events

Figure 11. Types of mRNA splicing

○ SE (Skipped Event): ~40%. When an entire specific exon is either included or excluded.

○ A5SS (Alternative 5’ Splice Site): ~7.9%. The splice junction at the 5’ end of the exon is used differently.

○ A3SS (Alternative 3’ Splice Site): ~18.4%. The splice junction at the 3’ end of the exon is used differently.

○ RI (Retained Intron): <5%. Introns that do not code for amino acid sequences are either retained or spliced.

○ MXE (Mutually Exclusive Exon): When one exon is included, the other is spliced out, and vice versa. This exclusive splicing is mainly observed in brain tissue.

○ Alternative Promoter

○ Alternative Poly-A

○ Note that the various types of hemoglobin are not due to alternative splicing but rather time-specific expression.

⑥ 6^th. Exon shuffling

○ A gene is formed with a new combination of exons due to crossover events between different genes.

⑦ 7^th. Trans-splicing

Figure 12. Cis/Trans splicing

○ Type 1. Cis-splicing: The process in which exons within a single pre-mRNA are joined through 5’ ss (splice site) to 3’ ss reactions, resulting in the formation of mRNA.

○ Type 2. Trans-splicing: The process in which two different pre-mRNAs are joined into a single mRNA molecule.

○ 2-1. The process in which the spliced leader (SL) from the 5’ tss (trans splice site) is joined with the pre-mRNA at the 3’ tss.

○ 2-2. Trans-splicing between different genes

○ 2-3. Trans-splicing within the same gene: The direction of the template may differ, or the origin may be maternal or paternal. Exon duplication is also possible.

○ SL trans-splicing process

○ Step 1. The 5’ tss of SL RNA binds to the 3’ tss of pre-mRNA.

○ Step 2. Mature mRNA contains an SL exon and a TMG cap at the 5’ end and is translated in the cytoplasm.

○ Step 3. As a result of splicing, a Y-shaped product (SL intron + pre-mRNA outron) is formed, resembling the lariat intron product generated in cis-splicing.

○ Step 4. Y-shaped product rapidly degrades.

Figure 13. SL trans-splicing process

⑧ 8^th. mRNA stability regulation

○ Longer mRNA lifespan enables more protein translation.

○ Prokaryotic mRNA has a lifespan of about 2 to 3 minutes.

○ Eukaryotic mRNA has a lifespan ranging from several days to several weeks.

○ Example 1. Hemoglobin mRNA remains intact and is not degraded as long as the red blood cell is alive.

○ Globin mRNA is highly expressed in red blood cells, accounting for about two-thirds of total blood RNA.

○ Example 2. Transferrin: An iron-importing and transport protein.

○ Iron Response Element (IRE): Located downstream of the coding region. An AT-rich sequence.

○ When Fe³⁺ increases: IRE-binding protein (IRE-BP) becomes inactive → The poly-A sequence of transferrin mRNA is removed → mRNA is degraded.

○ When Fe³⁺ decreases: IRE-BP becomes active → The poly-A sequence of transferrin mRNA is protected → mRNA is stabilized → Expression increases.

⑺ 5^th. rRNA, tRNA transcription

① rRNA is transcribed in the nucleolus by RNA polymerase I.

② mRNA and microRNA are transcribed in the nucleoplasm by RNA polymerase II.

③ tRNA is transcribed in the nucleoplasm by RNA polymerase III.

④ The promoter for rRNA is located far upstream of the gene.

⑤ The promoter for mRNA and microRNA is located close upstream of the gene.

⑥ The promoter for tRNA is located close downstream of the gene.

⑦ TBP (TATA-binding protein) is required for all.

⑻ 6^th. rRNA processing, tRNA processing

① rRNA processing

○ Type 1. 45S rDNA in the nucleolus → 18S rRNA + 5.8S rRNA + 28S rRNA

○ Type 2. Cleaving unmethylated bases

② tRNA processing: Slight cleavage of 3’ and 5’ from pre-tRNA.

⑼ Regulation of Transcription in Prokaryotes

① Regulatory elements

○ Common sequences in the promoter (P) and operator gene (O) (repressor binding site)

○ Activator binding site

○ Example: E. coli cAMP-CAP (Catabolite Activator Protein) complex recognition site

② Operon: A mechanism for coordinated regulation by transcribing multiple genes into a single mRNA.

③ Regulon: A mechanism where multiple operons are regulated by a single protein.

○ Example: Maltose regulon in E. coli.

④ Regulation by transcription repressors

○ When a repressor binds to the operator gene, it prevents RNA polymerase from binding to the promoter.

○ Inducible operon: In the presence of an inducer, the repressor bound to the operator is removed, allowing RNA polymerase to bind to the promoter and initiate transcription.

○ Example 1. Catabolic operon: Lactose operon (3 genes)

○ Repressor operon: In the absence of an inducer, the repressor bound to the operator is removed, enabling RNA polymerase to bind to the promoter and initiate transcription.

○ Example 1. Biosynthetic operon: Tryptophan operon

⑤ Regulation by transcription activators

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

① Summary

○ Inducible operon: The substance of interest finally promotes the expression of the operon.

○ Repressor operon: The substance of interest finally inhibits the expression of the operon.

○ Positive regulation: Regulation involving activators. In inducible operons, the activator is activated by the target molecule, while in repressor operons, the activator is inhibited by the target molecule.

○ Negative regulation: Regulation involving repressors. In inducible operons, the target molecule inhibits the repressor, while in repressor operons, the target molecule activates the repressor.

② Lactose operon (lac operon): Inducible operon

○ Composition of lac operon: (Regulator) - CAP binding site - Promoter - Operating region - lac Z - lac Y - lac A

Figure 14. Composition of lactose operon

○ Regulator (lac I): Sites that encode inhibitors. It is not included in the operon because it has its own promoter (constantly expressed).

○ Operating site

○ Gene 1. Beta-galactosidase (lac Z)

○ β-galactosidase: Lactate lyase. Converts lactose into allolactose.

○ lac Z actually produces the α peptide, which is the N-terminal part of β-galactosidase.

○ α complementation: The process by which the α peptide combines with another peptide from the host to form β-galactosidase.

○ Gene 2. Beta-galactosid permease (lac Y): Lactose transporter. Expressed on cell membrane.

○ Gene 3. Beta-galactoside transacetylase (lac A)

○ Beta-galactoside transacetylase removes by-products.

○ Negative regulation of lac operon

○ The lac operon repressor can bind to the operator site.

○ The lac operon repressor is produced by the lac I gene and inhibits RNA polymerase from binding to the promoter.

○ When the lac repressor binds to allolactose, a derivative of lactose, it can no longer bind to the operator site (lactose acts as an inducer).

○ Positive regulation of lac operon

○ Background: The promoter of the lac operon has low affinity for RNA polymerase, so transcription does not occur efficiently even in the absence of the repressor.

○ Adenylyl cyclase (AC) converts ATP into cAMP.

○ Catabolite Activator Protein (CAP) binds to cAMP to form a complex.

○ The CAP-cAMP complex facilitates the binding of RNA polymerase to the promoter.

○ Glucose can inhibit adenylyl cyclase through an allosteric inhibition site, preventing the above mechanism from functioning.

○ Result: Glucose, being more efficient, is prioritized over the operation of the lac operon, avoiding the unnecessary synthesis of enzymes from the lac operon.

○ The lac operon provides a mechanism for diauxic growth in media containing both glucose and lactose.

○ Glucose (+), Lactose (+): cAMP decreases → Positive regulation decreases → Lactose-degrading enzymes are not produced.

○ Glucose (+), Lactose (-): cAMP decreases → Positive regulation decreases → Lactose-degrading enzymes are not produced.

○ Glucose (-), Lactose (+): Allolactose increases → Repressor binding to the operator decreases → Lactose-degrading enzymes are produced.

○ Glucose (-), Lactose (-): Allolactose decreases → Repressor binding to the operator increases → Lactose-degrading enzymes are not produced.

○ Operons derived from the lac operon

○ tac operon

○ lac I^-: The repressor is defective and cannot bind to the operator site, resulting in constant activation.

○ lac I^S: Lactose cannot bind to the repressor, resulting in constant inactivation.

○ lac O^c: The operator site is defective, preventing the repressor from binding to the operator, resulting in constant activation.

○ lac Z^-: Encodes a defective lac Z gene. β-galactosidase is not expressed, regardless of operon activity.

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

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