Exam 2 (Ch. 7-11)

Evidence of DNA being genetic material

1. Griffith transformation in 1928 found that transforming principle existed

2. Avery and MacLeod and McCarthy experiments in 1944 determined DNA to be the transforming factor/principle

3. Hershey and chase experiments 1950 concluded DNA was the genetic material

DNA Synthesis needs …

1) Substrate → deoxynucleoside Triphosphates

• Needs 2 substrates present

• dGTP, dATP, dCTP, dTTP

2) A mechanism → Primer:template junction

• DNA is synthesized by extending the 3’ end of primer

• 3’ OH nucleophilic attack to the alpha phosphate on the nucleoside to add a base pair

Bacterial DNA replication

• Usually bidirectional, from a single origin of replication

• Characterized by an expansion around the origin of replication, forming a replication bubble

  • At end of replication bubble is a replication fork → replication is complete when the replication forks meet

Players involved in DNA synthesis

• Helicase: unwinds the double helix

• Topoisomerase: relaxes supercoiling of DNA

• Single-stranded binding protein, SSB: Prevents reannealing of separated strands

• Primase: synthesizes RNA primers

• DNA pol III: synthesizes DNA

Origin of replication

• Where DNA replication starts

• Have sequences that attract replication enzymes

• i..e in E.coli, origin of replication sequence = oriC

• In bacteria, have similar (conserved) but not identical sequences

Consensus sequences

The nucleotides found most often at each position of DNA in the conserved region

DNA pol III: Leading strand

One copy of pol III synthesizes one daughter strand continuously in the same direction as fork progression

DNA pol III: Lagging strand

The other copy of pol III elongates the daughter strand discontinuously in the opposing direction to fork progression, via short segments (Okazaki fragments)

What does DNA pol I do?

• Uses two activities to complete replication:

  • 1) It’s 5’-3’ exonuclease activity removes the RNA primers

  • 2) It’s 5’-3’ polymerase activity adds DNA nucleotides to the 3’ end of the DNA segment preceding the primer

What does DNA ligase do?

Seals the gap between the resulting DNA segments

DNA polymerase mechanisms to proofread functions and make sure it’s incorporating the right base at each site

1. Prevent wrong base from being incorporated via physical interaction

   1. “correct” base pairing has particular angles, so polymerase can recognize that angle

2. Remove erroneous base if one happens to be incorporated (proofreading)

   1. Incorrect base-pairing stalls the polymerase b/c -OH is misplaced for next incorporation → causes end of growing strand to ‘flip’ into 3’-5’ exonuclease domain → several bases are removed, after which polymerase tries again

Finishing DNA replication in Eukaryotes

• Use telomeres and telomerase

  • Telomeres: the strands of nucleotide sequences at the end of each chromosome that act as a buffer to protect the genetic material inside the chromosome (aka repetitive sequences at the ends of chromosomes)

    • so they tend to get shorter over time as we age

    • ensure that incomplete chromosome replication doesn’t affect vital genes

  • Telomerase: the enzyme that adds telomeres to the end of chromosomes to maintain chromosome length

There must be an intermediate between DNA and protein

• Proteins are assembled in the cytoplasm

• DNA is confined to the nucleus

• RNA is found in both & chemically similar to DNA

Transcription

The synthesis of single-stranded RNA molecule by RNA polymerase

Features of Transcription

• Similar to DNA replication chemically and enzymatically but instead uses RNA polymerase/instead of deoxyribonucleotides, the new strand is ribonucleotides

• RNA product is displaced nearly immediately from template

• Less accurate

• Not interested in the whole genome, only sections

• Not bidirectional (no tethered polymerase)

• Can initiate w/out a primer

RNA polymerase

• Catalyzes the addition of each ribonucleotide to 3’ end of the new strand, and forms phosphodiester bonds between nucleotides

  • Enzyme that catalyzes RNA synthesis

  • Synthesizes large # of transcripts from a single gene

  • The two DNA strands re-anneal

  • Less proofreading in transcription (vs replication that has more proofreading)

Nucleoside vs Nucleotide

• Nucleoside: Nitrogenous base linked to 5-C sugar (ribose or deoxyribose)

• Nucleotide: Nucleoside that has 1 or more phosphate groups attached

Features of RNA pol

• Bacteria: single RNA pol

• Eukaryotes: 3 RNA pol (we focus on Pol II)

• RNA polymerase core sigma unit for confirmation change

Messenger RNA (mRNA)

• Produced by protein-producing genes and is a short-lived intermediary between DNA and protein

• The only type of RNA that undergoes translation

• Transcription of it is often followed by post-transcriptional processing

Functional RNA

Don’t encode proteins, but instead perform functional roles as RNA itself

Function RNA (eukaryotes only): Transfer RNAs (tRNAs)

Are encoded in dozens of forms and are responsible for binding an amino acid and depositing it for inclusion into a growing protein chain

Function RNA (eukaryotes only): Ribosomal RNA (rRNa)

Combines w/ numerous proteins to form ribosomes

Function RNA (eukaryotes only): Small nuclear RNA (snRNA)

Various types is found in the nucleus of eukaryotes and plays a role in mRNA processing

Function RNA (eukaryotes only): Micro RNA (miRNA) & interfering RNA (siRNA)

Are active in plant and animal cells and are involved in posttranscriptional regulation of mRNA

Bacterial Transcription Process

1. Promoter recognition and ID

2. Initiation

3. Elongation

4. Termination

   1. Rho-dependent

   2. Rho-independent

Bacterial Gene Structure: Promoter

• Controls the access of RNA polymerase to the gene

• Is immediately upstream (5’) to the start of transcription, referred to as the +1 nucleotide

Bacterial Gene Structure: Coding Region

The portion that contains the information needed to synthesize the protein product

Bacterial Gene Structure: Termination Region

• Regulates cessation of transcription

• Is immediately downstream (3’) to the coding segment of the gene

  • Note: only one copy of the DNA that makes up the gene will be transcribed

Promoter recognition

• All RNA polymerases

  • composed of pentameric core enzyme that binds to sigma subunit → core enzyme can transcribe RNA from a DNA template, but cannot bind the promoter or initiate RNA synthesis without the sigma subunit

  • sigma subunit is what recognizes the promotor at -10 and -35

    • deviations from these sequences decreases binding strength, impacting whether or not transcription actually starts or the polymerase just falls off → cell uses transcription factor binding to turn on or off making the protein product

• Multisubunit enzymes

• Pincer shaped

Promoter

• A double-stranded DNA sequence that is the RNA polymerase’s binding site

  • Base pairs are disrupted

  • “Transcription bubble” forms

  • Promotes 5’ → 3’ direction

• Located a short distance upstream of the coding sequence, within a few nucleotides of +1

• RNA polymerase recognizes and binds to promoters by the presence of consensus sequences

Transcription Initiation

1. Binding to closed complex - ds DNA

   1. Promoter recognition by sigma subunit

2. Complex transition to open complex → base pairs “melt” forming “bubble”

   1. Initiation starts when ma polymerase binds and melts DNA “open” to read the template

3. Next we escape the promotor → start transcribing mRNA and building the first nucleotides

   1. sigma dissociates after a few nucleotides

Transcription Initiation Process Steps

1. Holoenzyme makes a loose attachment to the promoter sequence, then binds tightly to form the closed promoter complex

2. Holoenzyme unwinds about 18 bp of DNA around the -10 position to form the open promoter complex

Transcription Elongation

• Holoenzyme initiates RNA synthesis at the +1 site

• After 8-10 RNA nucleotides have been joined, sigma subunit dissociates from the core enzyme

• DNA is unwound ahead of the enzyme to maintain about 18 base pairs of unwound DNA'; the double helix re-forms behind the RNA polymerase

Transcription Termination

• When transcription of the gene is completed, the core enzyme dissociates from the DNA