Module 12: Nucleic Acids

3 components of a nucleotide?

A nitrogenous base, a sugar, and a phosphate group

2 types of nitrogenous bases

purines: adenine, guanine and pyrimidine: cytosine, uracil, thymine

What distinguishes a nucleotide from a nucleoside?

A nucleotide has a phosphate group; a nucleoside does not

where does the base attach to the sugar in nucleotides

Purine: n9 and pyrimidine: N1

What distinguishes ribose from deoxyribose?

Ribose has a 2’-OH group; deoxyribose has a 2’-H

Why is DNA more chemically stable than RNA?

DNA lacks the 2’-OH group, preventing base-catalyzed strand cleavage

Why does DNA use thymine instead of uracil?

To distinguish between natural uracil and uracil formed by cytosine delamination—ensures error correction

What does uracil delamination signal in DNA?

A mutation—should be replaced with cytosine

What are the key nitrogenous bases in DNA?

Adenine, thymine, cytosine, guanine

What type of sugar is in DNA?

2’-deoxyribose

What is the central dogma?

DNA → RNA → protein

What are phosphodiester bond?

Covalent linkages between the 3’-OH of one nucleotide and the 5’—phosphate of the next

What are the base-parking rules in DNA

A—T: 2 hydrogen bonds.G—C: 3 hydrogen bonds

What is the directionality of DNA strands?

Antiparallel; one strand runs 5’ → 3’, the other 3’ → 5’

What is non-covalent interactions stabilizes the double helix

Hydrogen bonding between base pairs. Base stacking via van der Waals interactions. Helical twist repulsion between phosphates

What is chromatin made of

DNA wrapped around histone proteins

What is nucleosome?

DNA wrapped around a histone octamer

What is the role of histone H1?

Stabilizes the DNA wrapped around the histone core

How is DNA accessed for transcription or replication?

Via histone tail modification (acetylation/methylation) that loosen chromatin

What sugar does RNA use?

Ribose (with 2’-OH)

Which base is found in RNA but not DNA?

Uracil replaces thymine

What is the general structure of RNA?

Single-stranded, but can form stem-loop and complex secondary structures

3 major classes of RNA?

mRNA: carries genetic code. tRNA: brings amino acids. rRNA: form ribosome core.

What are ribozymes?

Catalytic RNA molecules with enzymatic activity

What are small nuclear RNAs (snRNAs)?

RNAs involved in mRNA processing within the nucleus

What other roles can RNA play?

Gene expression regulation and programmed cell death

What are the components of a nucleotide?

A nitrogenous base, a sugar, and at least one phosphate group

Nucleoside

base + sugar

Nucleotide

base + sugar + phosphate

What are the purine and their sugar linkages?

Adenine (A) and Guanine (G); bond to sugar at N9

What are the pyrimidine and their sugar linkage?

Cytosine (C), Uracil (U, RNA only), Thymine (T, DNA only); bond at N1

What forms do U, T, G, and C take at pH 7?

U, T, G = keto. C = enol

Why does base tautomerization matter?

It affect hydrogen bonding potential (donor/acceptor)

Why do nucleic acid absorb UV light?

Due to aromaticity and electron-rich nitrogenous bases

What is the naming rule for nucleotide carbons?

Use primed numbers for the sugar atoms

What defines a 3’ vs. 5’ nucleotide?

Location of the phosphate group on the sugar

How are nucleotides joined in DNA/RNA?

Via phosphodiester bonds between 3’ and 5’ carbon atoms

What direction is nucleic acid sequence read?

5’ to 3’

Why are DNA and RNA polyanions?

Their phosphate groups are negatively charged at physiological pH

What indicates RNA vs. DNA in notation?

“p” = 5’ phosphate. “d” = deoxy. presence of T = DNA; U = DNA

Why does DNA use thymine instead of uracil?

To distinguish U formed from cytosine delamination—enable accurate repair

Why does DNA use thymine instead of uracil?

To distinguish U formed from cytosine amination—enables accurate repair

Why is DNA deoxy

The lack of 2’-OH increases chemical stability—important for genetic storage

Why is RNA more susceptible to hydrolysis?

2’-OH acts as a nucleophile in base-catalyzed cleavage

What are the step in RNA hydrolysis?

OH- deprotonates 2’-OH → forms O-. (2) O- attacks phosphate → backbone cleavage. (3) water hydrolyzes intermediate → mono-phosphate

What are Chargaff’s rule?

A = T. G= C.

Features of the Watson-Crick Model?

Antiparallel strands, right-handed helix, major and minor grooves, base pairing, and base stacking stabilize helix

What are the DNA forms and which is most common?

A-DNA, B-DNA, Z-DNA, B-form is most stable

What are palindromes and why are they important?

Self-complementary sequences; mark gene boundaries and restriction enzyme sites

What are histones and their core types?

DNA-binding proteins; H2A, H2B, H3, H4 (core); H1 (linker

Nucleosome

DNA wrapped around a histone octamer

What is the 30-nm fiber?

Second-level packaging from nucleosome coils

What are mini bands and chromosome fibers?

Higher-order DNA structure for tight packing into mitotic chromosomes

mRNA function and feature?

Carries genetic code from nucleus to cytoplasm; read 5’ to 3’

Difference between eukaryotic and prokaryotic mRNA

E =one protein/mRNA and Pro= multiple protein/mRNA

rRNA role and structure?

Structural and catalytic components of ribosomes

tRNA features?

Carries amino acids to ribosomes; 3’ end = CCA; complex step-loop structure

snRNA

splicing

regulatory RNAs

gene regulation, apoptosis

ribozymes

catalytic RNA

why do DNA and RNA migrate in gel electrophoresis?

Their negatively charged phosphate backbones drive movements toward positive cathode

How does agarose concentration affect separation?

0.7% is better for large fragments. 2% is better for small fragments

What is a restriction digest?

Cleavage of DNA using restriction enzymes, which recognize specific sequences and cut DNA

What are sticky ends

overhangs (either 3’ or 5’)

Blunt ends

cut at same point on both strands

What is restriction mapping used for?

Determining restriction sites and analyzing DNA differences

Single Digest

One restriction enzyme

Double Digest

Two enzymes — show relative position of sites

Southern Blotting

DNA fragments are transferred to a membrane, denatured, and hybridized with a radio labeled probe to detect specific sequences

What is the chain-terminator method?

Incorporation of ddNTPs during replication terminates chains, allowing sequences identification by fragment length

What is required in Sanger sequencing?

Template DNA, primer, DNA polymerase, dNTPs, and one ddNTP per reaction

How is sequence read from gel?

Bottom to top, 5’ to 3’ direction of the synthesized strand

What makes automated sequencing faster?

Fluorescent ddNTPs allow all reaction in one tube; laser read emitted color in a chromatograph

What does the chromatogram show?

Colored peaks; each peak = beak identity, size = incorporation amount

What is molecular cloning?

Inserting a gene into a vector and replicating it in host cells

What are essential features of a plasmid vector?

Multiple cloning site, antibiotic resistance gene, and origin of replication

What enables blue/white colony screening?

lacZ gene into vector. Insert present = no beta-galactosidase = white colonies. no insert = active enzyme = blue colonies

What are the 3 steps of PCR?

Denaturation, annealing, and extension

What is need for PCR?

Template DNA, DNA polymerase, Primer, and dNTPs

Forward Primer

Binds to non-coding strand

Reverse Primers

Binds to coding strand

What is in vitro mutagenesis?

Primer with mismatched bases are used in PCR to introduce point mutation into DNA

Recombinant DNA

DNA made by combining material from different sources

What are protein expression vectors used for?

To express human protein in host organism for therapeutic use

What is site-directed mutagenesis used for?

To study how amino acid changes affect protein structure nd function

What is a transgenic organism?

An organism with foreign DNA introduced into its genome

How are transgenic zebrafish used in research?

As models for human diseases, i.e., ethanol’s effects on neural development

Gene therapy

Correcting genetic disorders by introducing functional genes into patients

What is a limitation of gene therapy?

Random gene insertion can disrupt vital genes—new delivery methods are under development.