nucleic acids

DNA structure in terms of backbone and bases

Sugar-phosphate backbone made of deoxyribose and phosphate linked by 3'-5' phosphodiester bonds. Double helix with complementary bases held by hydrogen bonds (A-T has 2 bonds, G-C has 3 bonds). The helix is approximately 20 Å wide with adjacent bases separated by 3.4 Å.

What stabilizes the DNA helix

Base stacking through van der Waals forces between stacked bases, and hydrophobic interactions where bases are tucked inside and polar residues are outside.

Major and minor grooves in DNA

They exist because glycosidic bonds in base pairs aren't diametrically opposite. The major groove is wider while the minor groove is narrower. Both are lined with specific chemical groups that proteins can recognize.

Semi-conservative replication and Meselson-Stahl experiment

Each new DNA molecule contains one original strand and one newly synthesized strand. Meselson-Stahl labeled parent DNA with ¹⁵N (heavy), then replicated in ¹⁴N medium. After one replication, DNA was half-heavy with one ¹⁵N and one ¹⁴N strand. After two replications, half was half-heavy and half was fully light, proving semi-conservative replication.

Base pairing enables DNA replication

The sequence of one strand determines the sequence of the other. The two strands separate and act as templates, with new complementary strands synthesized using base-pairing rules where A pairs with T and G pairs with C.

Why DNA is long and what directionality means

E. coli genome is 4.6 million nucleotides. Directionality means DNA strands have 5' and 3' ends, and the sequence ACG is not the same as GCA because direction matters for reading genetic information.

DNA packaging in cells

DNA forms supercoiled structures, which compacts the long molecule into the nucleus or nucleoid region.

DNA polymerase III uses a primer and template in replication

Primase synthesizes an RNA primer of 10-12 nucleotides with a 3'-OH group. DNA polymerase III binds and extends from the 3'-OH, adding deoxyribonucleotides complementary to the template strand. New nucleotides are added only to the 3' end with base pairing ensuring accuracy.

Energy requirements for DNA replication

dGTP, dTTP, dCTP, and dATP are consumed and provide both the deoxyribonucleotide building blocks and the energy for polymerization. As each nucleotide is added, pyrophosphate is released, making the reaction exergonic.

Role of the 3'-5' exonuclease domain in DNA polymerase III

This proofreading activity removes incorrectly paired nucleotides immediately after incorporation. If a wrong base is added, the exonuclease cuts it out, allowing polymerase to add the correct nucleotide and reducing error rate from approximately 1/10⁴ to 1/10⁷ per base.

DNA strand elongation reaction

The 3'-OH of the growing strand attacks the α-phosphate of the incoming dNTP. A new phosphodiester bond forms and pyrophosphate leaves. The overall reaction is (DNA)ₙ + dNTP → (DNA)ₙ₊₁ + PPi.

Leading and lagging strands differ

The leading strand is synthesized continuously in the 5' to 3' direction in the same direction as replication fork movement, requiring only one primer. The lagging strand is synthesized discontinuously in Okazaki fragments of approximately 1000 nucleotides in prokaryotes and 100 nucleotides in eukaryotes, running 5' to 3' opposite to fork movement and requiring multiple primers.

Why Okazaki fragments are necessary on the lagging strand

DNA polymerase can only add nucleotides in the 5' to 3' direction and only to a 3'-OH group. At the replication fork, only one strand runs 5' to 3' (the leading strand). The lagging strand runs 3' to 5', so it must be synthesized discontinuously in short fragments going backwards relative to fork movement.

Origin of replication (oriC)

A special DNA region with specific features where replication begins. E. coli has one oriC while eukaryotes have many. The prepriming complex assembles at this location.

Role of helicase

Unwinds the double helix by breaking hydrogen bonds between base pairs to allow single strands to act as templates.

Role of SSB (single-strand binding proteins)

They keep the separated DNA strands from re-annealing or base pairing until they can be replicated.

Roles of DnaA and DnaB in E. coli replication initiation

DnaA binds to oriC and initiates the prepriming complex assembly. DnaB, which is helicase, unwinds the DNA at the origin.

Role of primase

Synthesizes short RNA primers of 10-12 nucleotides with a 3'-OH group needed to initiate DNA synthesis. It makes one primer on the leading strand but many primers on the lagging strand.

Role of DNA polymerase III

The main replicative enzyme with very high processivity, staying attached for approximately 1000 nucleotides on the leading strand and making short Okazaki fragments on the lagging strand. It adds nucleotides to the 3'-OH with 3'-5' exonuclease activity for proofreading.

Role of the sliding clamp in DNA replication

Increases the processivity of DNA polymerase III by keeping it attached to DNA longer. It is used on both the leading and lagging strands.

Role of DNA polymerase I in replication

Not required for continuous synthesis on the leading strand. On the lagging strand, it removes RNA primers using 5' to 3' exonuclease activity and fills in the gaps left behind.

Role of ligase

Seals the final nick in the DNA backbone by forming a phosphodiester bond between the 3'-OH and 5'-phosphate of adjacent fragments. It is required on the lagging strand to join Okazaki fragments.

Trombone model of DNA replication

Both leading and lagging strand synthesis happen at the replication fork simultaneously. The lagging strand loops out like a trombone slide, allowing DNA polymerase III to work on both strands at the same replication fork.

Four general ways DNA gets damaged

Oxidation through hydroxyl radicals, deamination through removal of amino groups, alkylation through addition of alkyl groups, and UV radiation causing thymine dimers.

Mutagens and agents that cause DNA damage

Mutagens are chemical agents that alter DNA bases. Agents include UV light, X-rays, hydroxyl radicals from oxidative stress, alkylating chemicals like aflatoxin B1, nitrous acid, and spontaneous hydrolysis.

Guanine oxidation (8-Oxoguanine)

A hydroxyl radical reacts with guanine to form 8-Oxoguanine. This form pairs with adenine instead of cytosine. After replication, one daughter DNA has G-C while the other has A-T, resulting in a mutation.

What happens during cytosine deamination

The amino group is removed, converting cytosine to uracil. Uracil does not belong in DNA and is recognized as damage.

Consequence of 5-methylcytosine deamination

5-methylcytosine, which is used for gene regulation, is deaminated to thymine. This is mutagenic because thymine is a normal DNA base and will not be recognized as damage.

What happens during adenine deamination

Adenine is deaminated to hypoxanthine. Hypoxanthine pairs with cytosine instead of thymine, causing mutations.

Guanine alkylation

An alkyl group, such as from aflatoxin B1 epoxide, is added to N-7 of guanine. This distorts the base and causes mispairing.

Thymine dimer

UV light covalently links adjacent pyrimidines, often thymine, along the DNA strand through a cyclobutane ring. This prevents base pairing and blocks replication and transcription.

Basic steps of any DNA repair mechanism

Recognition where damage is detected by repair enzymes, removal where damaged nucleotides are excised, replacement where DNA polymerase fills in the gap using the complementary strand as template, and ligation where DNA ligase seals the backbone.

Direct repair

Repair that occurs without removing fragments of DNA. Photolyase uses visible light energy to photochemically cleave the cyclobutane ring of thymine dimers, restoring the original bases.

Mismatch repair

MutS recognizes the mismatch and MutL binds and recruits MutH, an endonuclease. An exonuclease excises the incorrect region, DNA polymerase fills the gap, and ligase seals it. The cell recognizes which strand has the error using methylation patterns.

Nucleotide excision repair (NER)

It recognizes distortions in the helix. UvrABC excinuclease cuts DNA at two sites around the lesion. DNA polymerase fills the gap and ligase seals the backbone. It removes large bulky adducts.

Base excision repair (BER)

Glycosylase flips the defective base into its active site and cleaves the glycosidic bond. AP endonuclease nicks the phosphodiester backbone. Deoxyribose phosphatase removes the deoxyribose phosphate unit. DNA polymerase inserts the correct nucleotide. Ligase seals the strand. It removes single damaged bases.

Why DNA uses thymine instead of uracil

Cytosine spontaneously deaminates to uracil. In DNA, uracil is recognized as damage because it is not normal and is removed. In RNA, uracil is normal so this system would not work. Using thymine with its methyl group allows cells to distinguish normal from damage-derived bases.

Restriction endonucleases used in genetic engineering

They recognize and cut specific DNA sequences, often leaving sticky ends with single-stranded overhangs. To remove a sequence, cut on both sides and excise the fragment. To insert a sequence, cut target and source DNA with the same enzyme, mix to allow sticky ends to base pair, then ligase seals the nicks to create recombinant DNA.

DNA electrophoresis and how to interpret gels

DNA is loaded into wells at the gel's top. An electric field is applied and DNA, being negatively charged, migrates toward the positive electrode. Smaller fragments move faster and farther while larger fragments move slower and less far. Bands appear at positions corresponding to fragment sizes. Compare to a size standard ladder to identify specific sizes.

Three types of RNA and their functions

mRNA (messenger RNA) carries genetic information from DNA to the ribosome with variable length and temporary existence. rRNA (ribosomal RNA) is part of the ribosome's catalytic and structural machinery and is stable and abundant. tRNA (transfer RNA) brings amino acids to the ribosome and recognizes codons via its anticodon and is stable.

E. coli RNA polymerase and the role of sigma factor

The core enzyme (αα'ββ'ω) synthesizes RNA but cannot recognize promoters on its own. Sigma factor (σ) associates with the core to form the holoenzyme and guides polymerase to the -10 and -35 promoter boxes. After transcription starts, sigma dissociates and the core continues synthesis.

Three steps of RNA synthesis

Initiation where the polymerase-sigma complex binds at the promoter and synthesizes the first 8-10 nucleotides before sigma is released. Elongation where the core polymerase adds ribonucleotides complementary to the template strand in the 5' to 3' direction at approximately 40-50 nucleotides per second. Termination where polymerase stops at the terminator sequence and RNA is released.

Important sequences for transcription initiation and termination

For initiation at the promoter, the -35 box has consensus TTGACA and the -10 box or Pribnow box has consensus TATAAT. For termination, intrinsic terminators have a GC-rich inverted repeat in the RNA followed by a poly-U tract that forms a hairpin causing pause and dissociation, while rho-dependent terminators require the Rho protein.

How to translate an mRNA sequence into amino acids

Read mRNA in triplet codons of 3 nucleotides from 5' to 3'. Use the genetic code table to match each codon to its amino acid. The start codon is AUG (methionine) and stop codons are UAA, UAG, and UGA. For example, 5'-AUGCCGGUAAA-3' translates to Met-Pro-Val-Stop.

Roles of tRNA, mRNA, and rRNA in protein synthesis

mRNA carries genetic information and provides codons specifying which amino acid to add. tRNA brings the correct amino acid and its anticodon pairs with the mRNA codon. rRNA is part of the ribosome and catalyzes peptide bond formation, serving as the actual catalyst rather than the protein.

Role of aminoacyl-tRNA synthetase

It catalyzes tRNA charging with the reaction Amino acid + ATP + tRNA → Aminoacyl-tRNA + AMP + PPi. Each enzyme is specific for one amino acid and its cognate tRNA or tRNAs, maintaining fidelity of the genetic code.

Three steps of translation

initiation, elongation, termination

Energy cost of translation

2 GTP are used per aminoacyl-tRNA, one for EF-Tu delivery and one for EF-G translocation. 1 GTP is used for initiation and 1 GTP for termination. Additionally, ATP is used by aminoacyl-tRNA synthetase for tRNA charging. The typical total is approximately 4 GTP per amino acid, which is equivalent to approximately 4 ATP per peptide bond.

fMet-tRNA^fMet and why it is important

In prokaryotes, this is the initiator tRNA carrying N-formylmethionine with a formyl group added to methionine's amino group. Unlike other Met-tRNAs, it binds directly to the P site rather than the A site and is recognized by initiation factor IF2. It pairs with the start codon AUG. After the first peptide bond forms, the formyl group is usually removed. It is essential for proper initiation at the correct location.