DNA + transcription

What are DNA and RNA and what are their three parts

they are nucleotide polymers

made of phosphate group, 5-carbon sugar, and a nitrogenous base

How do sugars differ between DNA and RNA

RNA has a ribose sugar with an OH at the 2’ position. DNA has a deoxyribose sugar with an H at the 2’ position

Bases in RNA and DNA

Pyrimidine bases (one ring): Thymine (has a methyl group), Cytosine, Uracil (only in RNA)

Purine bases (double rings): Adenine, Guanine

In a nucleic acid, each base is attached to either a ribose or deoxyribose sugar 1’ position by the bond off of nitrogen on the bottom

Features of the 5’ and 3’ positions

Phosphate group attaches to the 5’ carbon, and nucleotides can differ in the number of attached phosphates (between 1 and 3 attached), and only nucleotide triphosphates can participate in polymerization

The 3’ carbon has a hydroxyl group

How are nucleotides polymerized

Nucleotides are linked together in condensation reactions to form a phosphodiester bond. This requires a free 3’ OH because the phosphate attached to the 5’ carbon covalent bonds to oxygen at the 3’, so it needs the OH to bond

Energy for this synthesis cleaves off 2 phosphate groups to make it an exergonic reaction. Therefore energy for DNA synthesis comes from the triphosphate group hydrolysis

Describe a nucleotide polymer

nucleotides are attached together into chains by a phosphodiester bond. All nucleotide polymerization in every setting occurs in a 5’→3’ direction, ie bases are never added to the 5’ end

The polymer has a 5’ and a 3’ end and DNA has an invariant phosphodiester backbone and a variant sequence of bases

characteristics of DNA structure

two strands of nucleotides in a double helix, strands oriented antiparallel, bases point inside the helix, opposing bases pair by hydrogen bonding. Only purine-pyrimidine pairs fit inside the double helix

complementary base pairing

A and T are held together by 2 hydrogen bonds (or A pairs with U in RNA)

G and C are held together by 3 hydrogen bonds. The pairs match in shape and chemistry, allowing the H-bonds

Chargaff’s rule about base pairing: [A]=[T] and [G]=[C]

DNA denaturation

heat denatures DNA because it breaks H-bonds - denatures dsDNA into ssDNA (double to single stranded)

As temp cools, single strands anneal forming dsDNA again. There is no DNA denaturation at body temp

How does DNA form a template for its own synthesis

if H-bonds between complementary base pairs are broken, the DNA helix can separate. Each strand of DNA can serve as a template for the formation of a new strand. Free nucleotides attach according to complementary base pairing. When new strands polymerize to form a sugar phosphate backbone, secondary structure is restored

Initiation of DNA replication

Bacterial chromosomes have a single point of origin. DNA separates at only one place

Eukaryotic chromosomes have multiple origins of replication. Replication proceeds in both directions from each starting point

What do DNA polymerases require

template strand that determines the identity and sequence of bases to be incorporated

an existing 3’ OH to add new nucleotides to

dNTPs, which are a combination of deoxyATP, dCTP, dTTP, and dGTP, which meas needs a pool of all 4 DNA monomers

Needs both a template and a primer with a 3’ OH group. Polymerase can’t synthesize a completely single or double stranded DNA. Needs single strand as a template plus 3’ end to start synthesis

basic features of DNA replication

new nucleotides are added to an existing chain by phosphodiester bonds. Catalyzed by DNA polymerase. Requires a template chain and dNTPs. Proceeds in 5’ to 3’ direction and added nucleotide creates a new 3’ OH

5 important enzymes

primase - catalyzes the synthesis of the RNA primer on an okazaki fragment

DNA polymerase III - extends an okazaki fragment and leading strand

Helicase - catalyzes the breaking of H-bonds between base pairs and the opening of the double helix

DNA polymerase I - removes the RNA primer and replaces it with DNA

DNA ligase - catalyzes the joining of okazaki fragments into a continuous strand

Synthesis of leading strand

DNA is opened by helicase, unwound, and primed by primase. Primase can make complementary RNA strand to DNA without the 3’ OH.

Because parental DNA strands are antiparallel and synthesis only occurs in one direction, one new strand is made continuously and one is made discontinuously. Leading strand: DNA pol III moves in same direction as helicase, lagging strand: DNA pol III moves in the opposite direction as helicase

Synthesis of lagging strand

Primase and DNA pol III synthesize repeated okazaki fragments. DNA pol III moves away from the replication fork until it runs into the next primer.

Primase lays down new primers on the lagging strand, and each side of the origin of repliaction has both leading and lagging strands

How does DNA pol I work

for each RNA primer linked to DNA, there is 3’ OH group next to it. This will be used by DNA pol I to extend off of and replaces the RNA with DNA. It removes ribonucleotides of RNA primer and replaces them with deoxyribonucleotides in 5’ to 3’ direction.

DNA pol I extends off the upstream 3’ OH on the okazaki fragment

Finally DNA ligase closes the gap in the sugar phosphate backbone and covalently links okazaki fragments together

What is transcription

DNA is used as a template to make an RNA polymer. It is initiated at a promoter sequence and ends at a terminator sequence. Synthesized 5’→3’. Both DNA strands can serve as templates for transcription

RNA polymerase

catalyzes formation of the phosphodiester bond between triphosphate ribonucleotides. Polymerization is driven by release of energy from the triphosphate (same as DNA). 5’ to 3’ growth antiparallel to DNA template.

RNA pol unwinds the DNA without helicase, initiates new strands without a primer; only requires a template and nucleotide triphosphates (NTPs). Does not require existing 3’OH

How does RNA synthesis work

incoming triphosphate ribonucleotides (NTPs) are accepted if they correctly base pair with the template DNA. The 3’OH of the growing strand attacks the high energy phosphate bond of the incoming ribonucleotide, providing energy to drive the reaction

differences between DNA and RNA polymerase

DNA: template, dNTPs (deoxy), 3’OH primer, ATGC, synthesis 5’ to 3’

RNA: template, NTPs, no primer, AGCU, synthesis 5’ to 3’

Three major transcription processes

initiation - RNA polymerase is recruited to the promoter and activated

elongation - synthesis of RNA from DNA template

termination - RNA pol disassociates from DNA and transcription ends

initiation of transcription

promoters are regions in the DNA immediately upstream of the transcription start site. The promoter is a segment of DNA with specific sequence. RNA pol is recruited to the promoter. Once recruited and activated, transcription occurs. RNA pol unwinds DNA and RNA synthesis begins. RNA pol is doing the synthesis

template vs nontemplate strand

template (noncoding) strand is what RNA poly uses to determine the RNA sequence. RNA is complementary to this strand.

The nontemplate (coding) strand tells the sequence of RNA because it is also complementary to the template strand

termination of transcription

RNA pol stops adding ribonucleotides and the preRNA chain is released. Occurs at a specific site - transcription termination site - for each transcription unit (gene)