18. Nucleic Acid Metabolism + Genetic Information Processing

What are the overall steps of DNA replication?

1. Initiation

2. Elongation

3. Termination

Why is DNA replication semiconservative, bidirectional, and semi-discontinuous?

• Semiconservative: 1 strand template + 1 strand new

• Bidirectional: 2 Replication forks working simultaneously

• Semi-discontinuous: Leading is continuous + Lagging is discontinuous

What is the replication fork?

Site where replicating DNA has initially separated and new nucleotides are being added

What is the function of helicase?

Open strands at Origin of Replication

What is the function of topoisomerase (Gyrase)?

Relieve strain/Change supercoiled state of DNA

What are the characteristics of DNA polymerases?

• Pair incoming dNTP bases with template bases

• Require primer + 3’-OH (cannot initiate DNA synthesis de novo)

• Extend nucleic acid ONLY 5’ → 3’

• Use dNTPs as energy source - pyrophosphate hydrolysis to build new phosphodiester linkages in new strand

Why does DNA replication require a RNA primer?

DNA synthesis cannot be made by itself, it needs a pre-existing nucleic acid 3’-OH strand

What is the process of synthesis for the leading strand of DNA?

• Read Parent Strand: 3’ → 5’

• Daughter Strand: 5’ → 3’

• Primase: 1 RNA primer

• DNA Polymerase III adds first dNTP

What is the process of synthesis for the lagging strand of DNA?

• Read Parent Strand: 5’ → 3’

• Daughter Strand: 3’ → 5’

• Primase: Intermittent RNA Primers

• DNA Polymerase III copies lagging strand in between RNA primers

• Okazaki Fragments: small pieces of DNA w/ RNA heads

How are RNA primers removed and DNA fragments joined together?

• 5’ → 3’ Exonuclease RNase H to remove primers

• DNA Polymerase I hydrolyze RNA head and polymerize DNA behind

What are the causes of DNA damage?

• Replication Errors

• UV-induced base alterations

• Strands breaks

• Covalent cross-linking of strands

Proofreading

• Done by DNA Polymerases

• Detect incorrect base pair immediately as DNA is synthesized

• Remove and replace correct nucleotide

Direct Repair

• DNA Photolyase breaks Pyr = Pyr dimers

• Use UV light energy to break bonds that hold dimer together

• Dimer: from UV radiation exciting Pyr to dimerize and cause error in replication

Mismatch Repair

• Mismatch recognition

• Excision of region w/ mismatch

• DNA Poly fills gap

• DNA Ligase seals

Base Excision Repair

• DNA glycosylase recognize + remove damaged base

• Leaves abasic (AP) site

• AP Endonuclease cut DNA backbone at AP site

• DNA Poly insert correct nucleotide

• DNA Ligase seals

Nucleotide Excision Repair

• Damage recognition by NER proteins

• Helicase unwinds region

• Dual incisions from endonuclease

• Removal of damaged fragment

• DNA Poly fills gap

• DNA Ligase seals

What are the 3 major types of RNA?

mRNA

• Contain message to encode protein

• Rapidly degraded by nuclease

tRNA

• Carry AA to translation (the ribosome)

• Very stable

rRNA

• Make up much of ribosome

• Very stable, majority of cellular RNA

Which histone modifications are required to start gene expression?

Modification (Acetylation) of Lys residues

• Looser conformation and more readily transcribed

• HAT: Histone Acetyl-Transferase

• HDAC: Histone De-Acetylase

What is the composition and role of a gene?

Encode RNA + Provide instruction + Regular RNA production + Determine Cellular function

• Promoter Region

• Transcription start site/+1 position

• Template Strand Read: 3’ → 5’

• RNA Produced 5’ → 3’

• RNA-Coding region

• Terminator sequence

• Upstream bases (-)

• Downstream bases (+)

What is the overall process of transcription?

1. RNA Polymerase bind to promoter

2. Initiation of polymerization

3. Chain elongation

4. Chain termination

What is the function of a promoter?

Start site that contains sequence for RNA Polymerase binding (TATA Box or Pribnow Box)

What are the roles of Eukaryotic transcription factors?

To form a large initiation complex with RNA Polymerase + other protein complexes to regulate when/where/how transcription occurs

How do distant enhancer and silencer sites affect transcription and initiation?

They bind to transcription factors and facilitate initiation/prevention of transcription

What are the main characteristics of RNA Polymerases?

Prokaryote have 1 + Eukaryote have 3 (for each RNA)

• Initiate nucleotide polymerization w/o primer

• Synthesize nucleotide 5’ → 3’

• Regulated by transcription factors and inducers/repressors

What is the reaction catalyzed by RNA polymerase?

Double-helix of DNA opening to form the transcription bubble

• Initiation of Ribonucleotide Polymerization

What are the two methods of transcription termination in Prokaryotes?

• Intrinsic Termination (without rho)

  • Near termination sequence G:C sequences form stem-loop/hairpin

  • Poly-A sequence cause weaker attractions → mRNA/DNA dissociation

• Termination (with rho)

  • Rho: helicase hexamer protein binds mRNA and hydrolyze ATP (helicase activity) down strand

  • RNA Polymerase pause at end → rho pulls mRNA out of polymerase

What are covalent modifications of RNA and their purposes in Eukaryotes?

• 5’ Capping: prevent exonuclease degradation + aid in translation initiation

• UTRs: 5’ end for ribosome binding | 3' end for termination of translation

• RNA Splicing: Remove introns + Join exons w/ spliceosome → allow alternative splicing

What is the advantage of splicing protein-coding genes in Eukaryotes?

It creates a functional protein + protein diversity

Why is genetic code universal, redundant, and unambiguous?

• Universal: All organisms share same set of codons that specify same amino acids

• Redundant: Most amino acids are coded by more than one codon

• Unambiguous: Each codon only code for 1 amino acid

What are the structural features of tRNAs?

Cloverleaf Secondary Structure: 4 Loops

• 3’-end = Amino Acid acceptor end

• Anticodon Loop binds mRNA codon

What are the substrates, products, and catalytic activities of aminoacyl-tRNA synthetases?

Substrates:

• Amino Acid + ATP +tRNA

Products:

• Aminoacyl-tRNA + AMP + PP_i

Catalytic Activity:

• Activation of Amino Acid

• Charging of tRNA

How does one tRNA anticodon pair with more than one mRNA codon?

Wobble Hypothesis:

• 3rd codon position allows “wobble”

• Flexible, non-standard base pairing → degeneracy

What is the importance of ribosomal RNA?

• Structural core of ribosome

• Catalyze peptide bond formation

• Ensure accurate alignment/movement during translation

What are the 3 tRNA binding sites in the ribosome?

• A site - Aminoacyl-tRNA arriving site

• P site - Peptidyl-tRNA site growing polypeptide chain

• E site - Exit site of empty tRNA after donating AA

What are the events of translation initiation, elongation, and termination?

• Initiation: Small subunit + mRNA + initiator tRNA assemble → then large subunit joins

• Elongation: tRNA entry → peptide bond → translocation

• Termination: Stop codon → release factors → peptide released → ribosome dissociates

What steps in translation are carried out by proteins vs. RNA?

Protein:

• Charge tRNA

• Start codon recognition

• Translocation

• Stop codon recognition

• Ribosome disassembly

RNA:

• Codon-anticodon pairing (tRNA + rRNA)

• Peptide bond formation (rRNA)

• Direct amino acids (mRNA

Why does transpeptidation (peptide bond formation) not require free energy input?

Peptide bond formation is driven by breaking of ester bond on aminoacyl-tRNA

How does the ribosome maximize the accuracy of translation?

• Proofreading codon-anticodon pairing at A site

• GTP proofreading

• Translocating 1 codon at a time

What is the role of GTP in translation?

GTP Hydrolysis allows release factors to dissociate

What is a polysome?

A single mRNA being translated simultaneously by multiple ribosomes

• AKA Polyribosome

What is the significance of post translational modification?

It allows proper protein folding, protein activation, protein regulation, correct protein transport, and degradation when needed