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 template + 1 new

• Bidirectional: 2 Replication forks

• Semi-discontinuous: Leading continuous + Lagging 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 dNTP bases with template

• Require primer + 3’-OH (no de novo)

• Extend nucleic acid ONLY 5’ → 3’

• Use dNTP (pyrophosphate hydrolysis) as energy source

Why does DNA replication require a RNA primer?

DNA synthesis cannot be made by itself, needs pre-existing 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 add dNTPs

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

• Read Parent Strand: 5’ → 3’

• Daughter Strand: 5’ → 3’

• Primase: Intermittent RNA Primers

• DNA Polymerase III copies lagging strand b/t RNA primers

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

How are RNA primers removed?

Exonuclease RNase H (5’ → 3’)

How are DNA fragments joined together?

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

DNA Polymerase I and III detect mispaired bases and use 3’ → 5’ exonuclease activity to remove incorrect nucleotide and resume synthesis

What is a dimer?

UV radiation exciting Pyr to dimerize and cause error in replication

Direct Repair

DNA Photolyase binds Pyr = Pyr dimers and use UV light energy to break bonds that hold dimer together

Mismatch Repair

Corrects point mutations and change single base pair caused by replication errors, recombination, and base deamination

Base Excision Repair

Acts on single base that has been modified by oxidative rxns

1. DNA glycosylase recognize + remove damaged base

2. Leaves abasic (AP) site

3. AP Endonuclease cut DNA backbone at AP site

4. DNA Poly insert correct nucleotide + DNA Ligase seals

Nucleotide Excision Repair

Recognize and repair larger regions of damaged DNA

1. Damage recognition by damage-binding proteins/RNA polymerase

2. Helicase opens up two strands

3. Endonuclease does incision and fragment released

4. DNA Poly fills gap + DNA Ligase seals

What are the 3 major types of RNA and what do they do?

mRNA: carry genetic information for protein synthesis (short-lived)

tRNA: deliver amino acids to ribosome (stable)

rRNA: make up much of ribosome/cellular RNA (very stable)

Which histone modifications are required to start gene expression?

Modification (Acetylation) of Lys residues

• Looser conformation and more readily transcribed

• HAT: Histone Acetyl-Transferase → Activate Transcription

• HDAC: Histone De-Acetylase → Repress Transcription

What is the composition and role of a gene?

• Regulatory region, +1 transcription start site, and RNA-coding region

• RNA synthesized 5’ → 3’ from DNA Template 3’ → 5’

• Upstream Sequences: regulate transcription inhibition

• Downstream Sequences: transcribed into RNA

What is the overall process of transcription?

1. RNA Polymerase bind 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?

Recognize/bind promoter DNA and assemble RNA polymerase II into transcription initiation complex to regulate transcription

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

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

What are the main characteristics of RNA Polymerases?

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

• Do NOT require primer

• Synthesize RNA 5’ → 3’

• Regulated by transcription factors + inducers/repressors

What reaction is catalyzed by RNA polymerase?

Formation of phosphodiester bonds between NTPs to produce RNA strand

What are the two methods of transcription termination in Prokaryotes?

• Intrinsic Termination (without rho)

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

• Termination (with rho)

  • Rho protein binds RNA and uses ATP to release RNA Poly

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

• 5’ capping: prevent exonuclease degradation + translation initiation + ribosome binding

• 3’ poly A tail: termination of translation

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

Splicing removes introns and enables alternative splicing → increase protein diversity and regulatory flexibility in eukaryotes

Why is genetic code universal, redundant, and unambiguous?

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

• Redundant: An amino acid can coded by more than one codon

• Unambiguous: Each codon specific for one 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 (form L-shape 3-D structure)

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

Amino Acid + ATP +tRNA → Aminoacyl-tRNA + AMP + PP_i

Catalytic Activity:

• Activation/Adenylation of Amino Acid

• Transfer tRNA

• Proofreading

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

Wobble Hypothesis:

• Some anticodons can recognize more than one codon at 3rd position

• 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:

• Deliver tRNA to A site

• Start/Stop codon recognition

• Translocation

• Ribosome disassembly

RNA:

• Codon-anticodon pairing + A, P, E site (tRNA)

• 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

What is the role of GTP in translation?

GTP Hydrolysis allows:

• tRNA delivery

• Ribosome translocation

• Termination (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

• Degradation when needed