DNA and RNA structure

• DNA possesses three essential features: stability with mutability, faithful replication, and the ability to carry information

Structure of DNA

• DNA is composed of deoxyribose, and the bases adenine (A), thymine (T), cytosine (C), and guanine (G)
• It is double-stranded and forms an antiparallel helix
• The 5' end has a phosphate group, and the 3' end has a hydroxyl (OH) group

Structure of RNA

• RNA contains ribose, and the bases adenine (A), uracil (U), cytosine (C), and guanine (G)
• It is single-stranded
• The 5' end has a phosphate group, and the 3' end has a hydroxyl (OH) group
• RNA can form stem-loop structures

Importance of 2' and 3' Hydroxyl Groups

• Hydroxyl groups are crucial for nucleic acid structure and function, particularly in RNA
• The 3' OH group is essential for DNA and RNA polymerase to add new nucleotides
• The 2' OH group contributes to the stability and structure of RNA

DNA Replication in Prokaryotes

• Prokaryotes use a single origin of replication because DNA is circular

DNA Replication in Eukaryotes

• Eukaryotes use multiple origins of replication due to larger genomes
• DNA is linear, requiring telomeres and telomerase to prevent the loss of genetic material
• Eukaryotic DNA replication is more complex, involving multiple polymerases and a highly regulated initiation process

Origin of Replication

• Prokaryotes have a single origin (OriC in bacteria)
• Eukaryotes have multiple origins per chromosome

Replication Bubble

• Prokaryotes have one replication bubble per circular chromosome
• Eukaryotes have multiple replication bubbles due to multiple origins

Replication Fork

• Prokaryotic replication forks move bidirectionally
• Eukaryotic replication forks also move bidirectionally but are more complex

Replisome

• Prokaryotic replisomes include DNA helicase, primase, DNA polymerase III, a sliding clamp, and topoisomerase
• Eukaryotic replisomes are more complex, including DNA polymerases α, δ, ε, helicase, primase, PCNA (sliding clamp), and topoisomerases

Telomeres

• Telomeres are absent in prokaryotes due to circular DNA, which prevents end loss
• Telomeres are present in eukaryotes and require telomerase to prevent end shortening of linear chromosomes

DNA Polymerases: Definitions and Functions

• DNA polymerases make phosphodiester bonds and require a template and a primer
• DNA Polymerase I removes RNA primers (via its 5' to 3' exonuclease activity), fills in gaps with DNA nucleotides (5' to 3' polymerase activity), and proofreads newly synthesized DNA (3' to 5' exonuclease activity)
• DNA Polymerase II functions as a backup enzyme for replication and DNA damage repair
• DNA Polymerase III is the primary enzyme for DNA replication, extending leading and lagging strands with high processivity (5' to 3' polymerase activity) and proofreading DNA (3' to 5' exonuclease activity)
• Poly A Polymerase does not need a primer but can only add As

Ligase

• Ligase makes phosphodiester bonds to repair gaps between nucleotides

RNA Polymerase Holoenzyme

• The RNA Polymerase Holoenzyme is a promoter enzyme in prokaryotic transcription
• It contains a σ (sigma) subunit that binds to the DNA promoter region, along with a core enzyme that reads the DNA from 3' to 5'

RNA Polymerases

• RNA polymerase I makes rRNA in transcription
• RNA polymerase II makes mRNA in transcription
• RNA polymerase III makes tRNA in transcription

mRNA

• mRNA carries information from DNA to mRNA, which passes information to proteins

rRNA

• rRNA is a component of ribosomes, essential for protein synthesis

tRNA

• tRNA molecules carry specific amino acids to the ribosome in translation
• Contains an anticodon that binds to the corresponding codon of mRNA
• Charged tRNA: A tRNA molecule with an amino acid attached to the 3' prime end (also called aminoacyl-tRNA)

miRNA

• miRNA is a functional RNA that regulates the amount of protein produced by eukaryotic genes

siRNA

• siRNA is a short double-stranded RNA produced by the cleavage of long double-stranded RNAs by dicer

snRNA

• snRNA is a short noncoding RNA found in the nucleus of eukaryotic cells that assists in RNA processing

Telomerase

• Telomerase uses RNA as a template to elongate the ends of eukaryotic chromosomes

Codon

• A codon is a three-nucleotide section of RNA that encodes a single amino acid

Anticodon

• An anticodon is a nucleotide triplet in tRNA that aligns with a particular codon in mRNA under the influence of a ribosome

Promoters

• Eukaryotic promoters are more complex, requiring multiple transcription factors and regulatory elements
• Prokaryotic promoters are simpler and directly recognized by RNA polymerase with the help of sigma factors

Transcription Initiation in Prokaryotes

• Occurs in the cytoplasm
• Promoters are needed for transcription
• Promoter: specific nucleotide sequence that allows transcription factors and enzymes (RNA polymerase holoenzyme) to bind to start the transcription process
• RNA polymerase holoenzyme contains a sigma subunit that binds to the DNA and a core enzyme that reads DNA from 3' to 5' and produces an RNA strand from 5' to 3'
• RNA polymerase holo can create any type of RNA needed in prokaryotic cells (i.e., mRNA, tRNA, sRNA, etc.)

Transcription Initiation in Eukaryotes

• Occurs in the nucleus
• RNA polymerase enzymes read the DNA and make new RNA strands
• RNA polymerase enzymes cannot function unless a transcription factor binds to the RNA polymerase enzyme and the promoter
• RNA polymerase I can only make rRNA which is used for ribosomes during translation

RNA Polymerase II

• RNA polymerase II will bind to the promoter via transcription factor to make mRNA and snRNA
  • mRNA is used for post-transcriptional modifications and translation
  • snRNA is used for splicing
• RNA polymerase III will bind to the promoter via transcription factor to make tRNA and a tiny bit of snRNA and sRNA
  • tRNA is used for the translation process

Initiation of mRNA Production

• Prokaryotic Promoter: Called "-35 Region", "-10 Region", "+1 Region"
• RNA Polymerase: Only contains RNA polymerase holoenzyme
• Eukaryotic Promoter: TATA Box, CAAT Box, GC Box
• RNA Polymerase: RNA pol II (mRNA)
• General Transcription Factors: TFIID

Elongation

• Prokaryotic RNA Polymerase: Reads the template strand (also called the anti-sense strand) from 3' → 5', synthesizes RNA from 5' → 3', opens the DNA, stabilizes ssDNA, and unwinds the DNA
• Eukaryotic RNA Polymerase: Reads the template strand (also called the anti-sense strand) from 3' → 5', synthesizes RNA from 5' → 3', opens the DNA, stabilizes ssDNA, and unwinds the DNA

Termination

• Prokaryotes: Termination occurs in two methods: Rho dependent and Rho-independent.
  • Rho dependent: As RNA polymerase makes the RNA, the Rho protein moves along the new RNA strand. Rho will cause RNA polymerase to break away -Rho-independent: As RNA polymerase is reading the template strand, it will encounter inverted repeats of nucleotides. Inverted repeats will then cause a hairpin loop. hairpin loop will then trigger the RNA polymerase to cleave itself off the new RNA strand
• Termination in Eukaryotes: As RNA polymerase is making the new RNA strand, it will encounter a polyadenylation signal (AAUAAA), triggering enzymes to come to the new RNA strand to cleave the RNA polymerase off the RNA strand

Post-transcriptional processing of rRNA and ribosome factory in the nucleolus

• rRNA Transcription: rRNA genes (rDNA) in the nucleolus are transcribed by RNA Polymerase I into a single precursor pre-rRNA
  • This pre-rRNA contains sequences for mature rRNAs that will form ribosomal subunits
• Pre-rRNA Processing
  • Cleavage: pre-rRNA is cut at specific sites to separate different rRNA components
  • Chemical Modifications: snORAs guide chemical modifications that enhance rRNA stability and function
  • Trimming & Maturation: Exonucleases refine rRNA segments to their final sizes
• Ribosome Assembly in the Nucleolus
  • rRNAs combine with ribosomal proteins, which are imported from the cytoplasm
  • Pre-ribosomal subunits (large & small) form inside the nucleolus
  • Subunits are exported to the cytoplasm, where they undergo final maturation and assemble into functional ribosomes for protein synthesis
  • The nucleolus functions as a "ribosome factory" for rRNA processing and ribosome assembly before ribosomes are used for translation

Elongation in Eukaryotes

• As RNA Polymerase II moves along the DNA, it elongates the mRNA transcript while simultaneously undergoing co-transcriptional processing to ensure the mRNA is stable and functional
  • The C-terminal domain (CTD) of RNA Polymerase II plays a crucial role in coordinating these processes
  • Shortly after transcription initiation, the 5' cap is added by the capping enzyme complex (CE), protecting mRNA from degradation and helps in ribosome recognition for translation

Intron Splicing

• During elongation, before transcription ends, the spliceosome (complex of snRNPs) recognizes splice sites
• Introns are removed, and exons are joined together
• Removes non-coding introns and enables alternative splicing, increasing protein diversity

Polyadenylation (Poly(A) Tail Addition)

• At transcription termination, cleavage factors recognize the polyadenylation signal (AAUAAA) and cleave the pre-mRNA
• Poly(A) polymerase adds ~200 adenosine residues to the 3' end
• Protects mRNA from degradation and enhances translation efficiency

Termination: Allosteric Model

• After the polyadenylation signal (PAS) is transcribed, transcription factors and elongation factors dissociate from RNA Polymerase II
• This causes a conformational change (allosteric effect) in RNA Pol II, reducing its processivity
• The weakened polymerase eventually detaches from the DNA template, leading to termination

Termination: Torpedo Model

• After the polyadenylation signal is cleaved from the pre-mRNA, the remaining downstream RNA remains attached to RNA Pol II
• Xrn2 (5' to 3' exonuclease) rapidly degrades this leftover RNA
• As Xrn2 catches up to RNA Pol II, it physically displaces the polymerase, forcing termination
• Both models may act together to ensure efficient termination, with allosteric weakening of RNA Pol II making it easier for the torpedo exonuclease to finish the job

rDNA genes, nucleolus post-transcriptional processing

• Transcription of rDNA: Ribosomal DNA (rDNA) genes in the nucleolus are transcribed by RNA Polymerase I, producing a large precursor rRNA (pre-rRNA)
• Pre-rRNA Processing: pre-rRNA undergoes cleavage at specific sites to separate the different rRNA components
• Chemical modifications are guided by small nucleolar RNAs (snoRNAs) to enhance rRNA stability and function

Exonuclease Trimming

• Exonucleases further refine the rRNA segments to their mature forms
• Ribosomal Assembly: The processed rRNAs combine with ribosomal proteins to form ribosomal subunits in the nucleolus

RNA Decay in Prokaryotes

• Initiation: Pyrophosphohydrolase (RppH) removes the 5' triphosphate from mRNA, converting it into a monophosphate, making it more susceptible to degradation
• Endonucleolytic Cleavage: RNase E, a key endoribonuclease, cuts the mRNA at internal sites, generating fragments with accessible 5' monophosphate ends
• Exonucleolytic Degradation: Exonucleases (e.g., PNPase, RNase R, RNase II) degrade the RNA fragments from either the 3' or 5' end, completing the decay process

RNA Decay in Eukaryotes

• Deadenylation: The Deadenylase complex (CCR4-NOT, PAN2-PAN3) removes the poly(A) tail, destabilizing the mRNA
• Decapping: The DCP1/DCP2 complex removes the 5' cap, exposing the mRNA for degradation
• 5' to 3' Degradation: The exonuclease Xrn1 rapidly degrades the mRNA from the 5' end after decapping
• 3' to 5' Degradation: (alternative pathway): exosome degrades mRNA from the 3' end, with DcpS breaking down the remaining cap structure

RNA Interference (RNAi)

• A natural process in eukaryotes that silences genes by degrading mRNA
• This is a post-transcriptional process that occurs after mRNA is synthesized from DNA
• Evolution provides cells more control over gene expression and act as defense mechanism against viruses that try to insert their genetic material into cells

Process of RNAi

• Double-stranded RNA (dsRNA) is processed into small interfering RNA (siRNA) by the enzyme Dicer.
• The siRNAs are incorporated into an RNA-induced silencing complex (RISC)
• The RISC finds and cleaves the target mRNA

RNAi Contributors

• dsRNA: Double-stranded RNA is a molecule that can trigger RNA interference (RNAi), a cellular mechanism for gene silencing
• Dicer: Dicer is an enzyme that acts as a molecular "scissor" for dsRNA, processing it into smaller fragments called siRNAs
• siRNA: Small interfering RNAs are short, double-stranded RNA molecules that are generated from dsRNA by Dicer
• Argonaute (ARGO): Argonaute proteins are a family of proteins that are essential for RNAi, and they bind to siRNAs to form the RISC complex
• RISC: The RNA-induced silencing complex (RISC) is a protein complex that includes an Argonaute protein and a siRNA, responsible for targeting and silencing mRNA
• Target mRNA: The siRNA, within the RISC complex, guides the complex to a specific mRNA molecule that is complementary to the siRNA sequence

Translation

• Translation: initiation, elongation, termination
• Protein translation in prokaryotes: ribosome built at ATG
• Protein translation in eukaryotes: ribosome built at 5' cap

Termination

• Stop codons recognized by release factors, not tRNA
• Translation terminates when the ribosome reaches a stop codon (UAA, UAG, or UGA) on the mRNA
• Unlike sense codons, stop codons do not have a corresponding tRNA
• Recognized by release factors (RFs)

DNA Polymerases (Used for replication)

• DNA Pol III in prokaryotes
• DNA Pol α, δ, and ɛ in eukaryotes

RNA Polymerases (Used for transcription)

• Single RNA Pol in prokaryotes
• RNA Pol I, II, III in eukaryotes

Functions

• Endonuclease: Cuts within a DNA or RNA strand by breaking internal phosphodiester bonds. Used in DNA repair, restriction enzyme activity, and splicing
• Exonuclease: Removes nucleotides one at a time from the ends of a DNA or RNA strand. Functions in proofreading, RNA degradation, and removal of primers
• Ligase: Seals nicks in the DNA backbone by forming phosphodiester bonds; essential in Okazaki fragment joining (lagging strand synthesis) and DNA repair
• Polymerase: Synthesizes new DNA or RNA strands by adding nucleotides in a 5' to 3' direction
• Different types: DNA Polymerase (replication & repair) and RNA Polymerase (transcription)
• Telomerase: Extends telomeres at the ends of eukaryotic chromosomes; uses an RNA template to add repetitive sequences, preventing DNA loss during replication
• Polyadenylase (Poly(A) Polymerase): Adds the poly(A) tail to mRNA post-transcriptionally. Enhances mRNA stability, nuclear export, and translation efficiency

Posttranscriptional modification of RNA

• RNA Editing: A-to-I (Adenosine to Inosine Editing)
  • Mechanism: Enzyme ADAR (Adenosine Deaminase Acting on RNA) converts adenosine (A) → inosine (I) in RNA
  • Effects: Alters codons, potentially changing amino acid sequences in proteins that can affect mRNA stability and splicing
• Methylation of RNA
  • Mechanism: Enzymes such as METTL3 add m6A (N6-methyladenosine) to RNA.
  • Effects: Regulates mRNA stability, translation efficiency, and splicing. Also plays a role in gene expression control

Posttranslational Modification of Proteins

• Phosphorylation: Addition of a phosphate group (PO4³¯) to serine (S), threonine (T), or tyrosine (Y) residues by kinases; removal by phosphatases. Regulates enzyme activity (activation/inactivation), controls cell signaling pathways
• Ubiquitination: Ubiquitin, a small protein, is attached to lysine residues of target proteins by ubiquitin ligases. Marks proteins for degradation via the proteasome, regulates protein stability and function, involved in DNA repair, transcription regulation, and immune responses

Ribosome functions as a ribozyme

• The ribosome functions as a ribozyme because it catalyzes peptide bond formation without requiring a protein enzyme
• The rRNA (ribosomal RNA) itself has enzymatic activity
• The large ribosomal subunit contains peptidyl transferase activity, which is crucial for forming peptide bonds between amino acids during translation
• The ribosome positions tRNAs in the P and A sites and catalyzes the transfer of the growing polypeptide from the P-site tRNA to the amino acid on the A-site tRNA

EPA sites in ribosome

• A Site (Aminoacyl-tRNA site): Entry site for charged tRNA carrying an amino acid. The anticodon of the tRNA binds to the complementary codon on the mRNA
• P Site (Peptidyl-tRNA site): Holds the tRNA carrying the growing polypeptide chain. The amino acid from the A-site tRNA is transferred and linked to the growing chain in the P site via a peptide bond
• E Site (Exit site): tRNA exits the ribosome after donating its amino acid. The now uncharged tRNA is released to be recharged with another amino acid

Redundancy of Genetic Code & Wobble Hypothesis

• The genetic code is redundant (degenerate): multiple codons can encode the same amino acid
• Redundancy ensures stability in protein synthesis and allows some flexibility in base-pairing during translation
• Proposed by Francis Crick: explains why a single tRNA can recognize multiple codons for the same amino acid
  • The third base in a codon (3' position on mRNA) can form non-standard base pairing with the first base of the anticodon in tRNA
  • A single tRNA can recognize multiple codons, reducing the number of tRNAs needed
• Wobble base pairing rules: G in the anticodon can pair with C or U in the codon, U in the anticodon can pair with A or G in the codon

Nonsense Mutations in mRNA

• Occurs when a codon for an amino acid is changed into a stop codon (UAA, UAG, or UGA), causing premature termination of translation and leading to truncated, nonfunctional proteins
• Nonsense Suppressor Mutations in tRNA: Certain tRNA mutations can suppress the effects of nonsense mutations, allowing translation to continue
• Mutant tRNAs carry an altered anticodon that can recognize the premature stop codon and insert an amino acid instead

Positive Gene Regulation

• Requires an Activator Protein: An activator protein binds to DNA and enhances transcription by helping RNA polymerase bind efficiently to the promoter. Increases gene expression when needed (e.g., CAP +cAMP, Arabinose, and AraC)

Negative Gene Regulation

• Requires a Repressor Protein: A repressor protein binds to the operator region, blocking RNA polymerase and preventing transcription. Turns genes off when they are not needed (e.g., lactose + I gene repressor)
• Regulatory proteins act as environmental sensors by changing shape when bound to effectors. This shape change modifies their ability to bind DNA, either activating or repressing transcription

Attenuation

• Regulates transcription by using translation to "sense" level of product, changing the 3D shape of the initial transcript
• Attenuation links transcription and translation to regulate gene expression mRNA folding changes based on ribosome stalling, determining whether transcription continues
• Example: Trp operon uses attenuation to prevent unnecessary tryptophan biosynthesis when levels are high
• Occurs only in prokaryotes, since transcription and translation are coupled

Untranslated Regions (UTRs) (RNA)

• Location: Non-coding regions of mRNA, located at the 5' and 3' ends of the coding sequence
• Function: -UTRs play a role in mRNA stability, translation efficiency, and mRNA localization
• Key sequences -5' UTR: Shine-Dalgarno sequence (prokaryotes): A sequence in the 5' UTR that facilitates ribosome binding -Kozak sequence (eukaryotes): A sequence in the 5' UTR that helps in translation initiation
• 3' UTR -Binding sites for regulatory proteins: The 3' UTR can contain sequences that bind to regulatory proteins, microRNAs (miRNAs), and other small RNAs, influencing mRNA stability and translation -Polyadenylation signal: A sequence in the 3' UTR that signals the addition of a poly(A) tail to the mRNA, which is important for mRNA stability

UTR Regions

• 5' UTR (leader sequence): The region at the 5' end of the mRNA, before the start codon
• 3' UTR (trailer sequence): The region at the 3' end of the mRNA, after the stop codon

Transcription: Bacteria vs Eukaryotes

• Bacteria
  • 500-700 genes -One RNA polymerase
  • Transcription and translation are coupled
• Eukaryotes
  • 16,000 - 24,000 genes -Three RNA polymerases
  • Transcription in the nucleus
  • Message processed for trip to the cytoplasm
• Eukaryotes: Three RNA polymerases
  • RNA I: Transcribes most rRNAs: 18s, 5.8s, 28s -RNA II TATA: Transcribes mRNAs and miRNAs -RNA III: Transcribes 5S rRNA, tRNAs, snRNAs, and III function
• Helicase and topoisomerase are needed