DNA Replication and Transcription

• Lecture 14 covers DNA Replication and Transcription to mRNA.

Learning Objectives

• Understand the central dogma of molecular biology.
• Know that DNA carries the instructions to make an organism.
• Be able to sketch the basic structure of a DNA double helix.
• Know the location of the 5' and 3' ends of a DNA strand.
• Understand the base pairing rules in DNA.
• Know that DNA polymerase function requires a template and a primer.
• Know how DNA polymerase can correct incorrectly formed base pairs.
• Sketch a bacterial chromosome during replication, identifying replication forks, leading and lagging strands, and the 5' and 3' ends at the replication fork.
• Know the functions of Helicase, Primase, DNA Polymerase III, Gyrase, DNA Polymerase I, and Ligase in DNA replication.
• Know the major differences between DNA polymerase and RNA polymerase.
• Understand how a bacterial cell knows where to begin transcribing a gene into mRNA.
• Know why some promoters are recognized by RNA polymerase better than others.
• Understand how bacteria use alternate sigma factors to regulate multiple genes simultaneously.
• Given a double-stranded DNA sequence and a promoter, identify the template strand.

Vocabulary

• Central Dogma: Describes the flow of genetic information from DNA to RNA to protein.
• Replication: The process of duplicating DNA.
• Transcription: The process of copying DNA into RNA.
• Reverse Transcription: Copying RNA into DNA.
• Translation: The process of synthesizing protein from RNA.
• Antiparallel double helix: DNA consists of two strands oriented in opposite directions.
• Hydrogen-bonded base pair: Complementary bases (A-T, G-C) held together by hydrogen bonds.
• Phosphodiester bond: The bond linking nucleotides in a DNA or RNA strand.
• 5' phosphate: The end of a DNA or RNA strand with a phosphate group attached to the 5' carbon of the sugar.
• 3' hydroxyl: The end of a DNA or RNA strand with a hydroxyl group attached to the 3' carbon of the sugar.
• Pyrophosphate: A molecule containing two phosphate groups, released during DNA synthesis.
• Primer: A short sequence of nucleotides used to start DNA synthesis.
• Template: The strand of DNA used to guide the synthesis of a new complementary strand.
• Proofreading: The ability of DNA polymerase to correct errors during DNA replication.
• Semiconservative: DNA replication where each new DNA molecule consists of one original and one new strand.
• Bidirectional: Replication proceeds in both directions from the origin.
• Theta replication: A mode of DNA replication common in circular chromosomes.
• Replication bubble: The structure formed during DNA replication with two replication forks.
• oriC/ter: The origin and termination site of replication.
• Leading strand: The strand of DNA that is synthesized continuously during replication.
• Lagging strand: The strand of DNA that is synthesized discontinuously in Okazaki fragments.
• Okazaki fragment: Short DNA fragments synthesized on the lagging strand.
• Helicase: An enzyme that unwinds the DNA double helix.
• Gyrase: An enzyme that relieves the tension created by unwinding DNA.
• Primase: An enzyme that synthesizes RNA primers to initiate DNA replication.
• Ligase: An enzyme that joins DNA fragments together.
• DNA polymerase III: The main enzyme responsible for DNA replication.
• DNA polymerase I: The enzyme that removes RNA primers and replaces them with DNA.
• RNA polymerase: The enzyme responsible for transcribing DNA into RNA.
• Sigma factor (sigma subunit): A protein that helps RNA polymerase bind to the promoter.
• Core enzyme: The part of RNA polymerase that carries out the synthesis of RNA.
• Promoter: A DNA sequence where RNA polymerase binds to initiate transcription.
• Consensus sequence: A sequence of DNA that represents the most common nucleotides at each position in a group of related sequences.
• -10/-35 sites: Regions in the promoter sequence recognized by the sigma factor.
• Gene: A segment of DNA that codes for a functional product, such as a protein.
• Template strand/nontemplate strand: The template strand is used to synthesize RNA, while the nontemplate strand has the same sequence as the RNA (except T instead of U).
• Hairpin loop terminator: A structure formed in RNA that signals the end of transcription.

Central Dogma of Biology

• Explains the flow of information:
  • DNA is replicated to make more DNA.
  • DNA is transcribed into RNA.
  • RNA is translated into protein.
• Transcription is carried out by RNA polymerase.
• Translation is performed on ribosomes.
• Replication is carried out by DNA polymerase.
• Reverse transcriptase copies RNA into DNA.

Nucleic Acids - Nucleotides

• Nucleotides consist of:
  • A sugar: deoxyribose (in DNA).
  • A phosphate group.
  • A nitrogenous base: either a purine (adenine, guanine) or a pyrimidine (cytosine, thymine).

Bases: Purine vs. Pyrimidine

• Purines: Adenine (A) and Guanine (G) possess a double-ring structure.
• Pyrimidines: Cytosine (C) and Thymine (T) possess a single-ring structure.

DNA Synthesis

• Occurs by adding nucleotides to the 3' hydroxyl (OH) group of the existing strand.
• A phosphodiester bond is formed between the 3' OH of one nucleotide and the 5' phosphate of the next.

DNA Structure

• Double stranded helix (2 strands).
• Antiparallel: One strand runs 5' to 3', and the other runs 3' to 5'.
• Complementary strands: A pairs with T, and G pairs with C.

DNA Strands Are Held Together By Hydrogen Bonds

• G always pairs with C.
• A always pairs with T.
• G:C pairs are stronger than A:T pairs due to having three hydrogen bonds vs two.

DNA synthesis by DNA polymerase

• DNA polymerase uses a template strand to synthesize the new strand.
• Synthesis occurs in the 5' to 3' direction.

Main Points

• DNA polymerase needs a template to copy.
  • It reads the template 3' to 5'.
  • It makes new DNA 5' to 3'.
• DNA polymerase adds new nucleotides to the existing 3'-OH (supplied by the sugar).
  • The existing 3'-OH attached to the existing nucleic acid polymer is called a primer.
• Energy for DNA synthesis comes from splitting pyrophosphate (P-P) from nucleoside triphosphates.
• In proofreading, if the wrong base is added, the hydrogen bond is made poorly, and the base is removed by DNA polymerase.

DNA Replication

• Semiconservative: After replication, each double helix contains one original and one new DNA strand.

DNA replication

• Semiconservative and bidirectional
• Begins at a specific sequence called the origin (oriC).
• Proceeds in both directions.
• Each new duplex has one strand which is a new copy and another of which is the template.
• Ends at a specific sequence 180° away around the circle called (ter).

DNA Replication (Step 1)

• DNA synthesis starts at the origin of replication (oriC) on the chromosome.
• DNA helicase unwinds the DNA, and the strands separate.

DNA Replication (Step 2)

• Primase (an RNA polymerase) synthesizes a short RNA primer complementary to the unwound region of DNA, providing a starting point for DNA polymerase.

DNA Replication (Step 3)

• DNA polymerase III catalyzes the synthesis of the new DNA.
  • It is guided by complementary base pairing between incoming nucleotides and the old DNA strand.

Replication

• DNA polymerase III has proofreading abilities.
  • It will back up and remove the incorrect nucleotide if an incorrect nucleotide is inserted then resynthesizes the DNA.
• Both strands replicate at the fork in the same direction, requiring one strand to loop back on itself.

Overview Showing Leading and Lagging Strand Orientation

• Okazaki fragments are synthesized on the lagging strand.
• RNA primers initiate synthesis on both the leading and lagging strands.

DNA Replication (Step 4)

• As synthesis progresses, the tension increases in the double helix ahead of the replication fork.
• DNA gyrase removes the tension, allowing unwinding to continue.

DNA Replication (Step 5)

• DNA polymerase I removes the RNA primer and replaces it with DNA.
• DNA ligase seals the gaps on each strand.
• Cell division occurs after synthesis is complete so that each new cell gets one copy of DNA.

Genes

• Genes consist of DNA information that codes for proteins.
  • Also includes coding sequences and regulatory (control) regions.

DNA is transcribed into RNA

• The minus strand serves as the template.
• RNA polymerase does not need a primer.
• In RNA, A pairs with U instead of A with T.

NOT ALL OF THE DNA IS TRANSCRIBED!!

• Only genes are transcribed into mRNA.
• Genes are located throughout the DNA on both strands.
  • Transcription starts at a special DNA sequence called the promoter.
• E. coli has 4,000,000 base pairs and about 4,000 genes.
  • Each gene in bacteria is about 1,000 base pairs.

Transcription (RNA Synthesis)

• RNA polymerase is the enzyme responsible for RNA synthesis.
  • In prokaryotes, one enzyme synthesizes all RNA molecules.
  • In eukaryotes, 3 enzymes exist:
    • RNA polymerase I (rRNA).
    • RNA polymerase II (mRNA).
    • RNA polymerase III (tRNA).

RNA polymerase

• The sigma subunit binds to the promoter sequence.
• Promoter is two consensus sequences separated by 15-17 base pairs.

Specific Amino Acids Binding

• Specific amino acids in the sigma subunit bind to specific nucleotides in the promoter sequence.
• The promoter sequence has complementary base pairs with nucleotides in the template strand and is on the non-template strand.

Promoter Strength

• Promoter sequence matches the consensus binding sequence for the DNA and sigma subunits. The closer it is, the stronger the promoter.
• Different sigma factors recognize different promoter sequences to allow transcription of different sets of genes.

Promoter Direction

• Transcription orientation dictates which DNA strand is used as a template.
• Transcription is 5' to 3' (like in DNA.)
• Therefore, a template is read 3' to 5'.

Termination

• When RNA polymerase encounters a terminator.
  • It will fall off the template and release the newly synthesized RNA.
• Termination is typically signaled by a hairpin loop.
• Promoter signals beginning of gene and loop signals end of gene.