Nucleic Acids & Gene Expression

Nucleotides

Monomers of nucleic acids made of C, H, O, N, and P atoms; also called nucleoside phosphates

Main components of a nucleotide

Pentose sugar, phosphate group, nitrogenous base

Most common sugar types in nucleotides

2-deoxy-D-ribose and D-ribose (2'-OH)

Purine bases

Adenine and Guanine, double ring (9C) structure

Pyrimidine bases

Cytosine, Thymine, and Uracil, Single ring (6C)

Phosphorylation states of nucleotides

Phosphate → ADP → ATP (via phosphoric anhydride linkages and dehydration synthesis)

Nucleoside

Nitrogenous base + sugar linked by glycosidic bond

Role of sugar in nucleosides

Increases water solubility compared to free bases

Nucleoside naming conventions

-idine for pyrimidines, -osine for purines

Adenine nucleoside

Adenosine

Adenine nucleotide

Adenosine-5'-phosphate or Adenylic acid

Guanine nucleoside

Guanosine

Guanine nucleotide

Guanosine-5'-phosphate or Guanylic acid

Cytosine nucleoside

Cytidine

Cytosine nucleotide

Cytidine-5'-phosphate

Thymine nucleoside

Thymidine

Thymine nucleotide

Thymidine-5'-phosphate or Thymidilic acid

Uracil nucleoside

Uridine

Uracil nucleotide

Uridine-5'-phosphate or Uridylic acid

Nucleic acid

Linear polymer of nucleotides joined by 3'-5' phosphodiester bonds

Phosphodiester bond formation

Phosphate of one nucleotide bonds to sugar of the next; water is released

Reason DNA uses thymine instead of uracil

Allows repair enzymes to detect deaminated cytosine (uracil) as a mutation

Reason DNA uses deoxyribose

Lacking 2'-OH increases stability and reduces hydrolysis

DNA vs RNA

Double-stranded vs Single-stranded

2-deoxy-D-ribose (2'-H) vs D-ribose (2'-OH)

Thymine vs Uracil

DNA helix twist

Right-handed, ~10-15 bases per turn

one complete turn 34 A

long base pairs are 3.4 A apart in B-DNA

diameter outside 20-21 A, inside 11 A

DNA strands orientation

Antiparallel

Base pairing in DNA

A-T via 2 H-bonds, G-C via 3 H-bonds

Stabilizing factors in DNA

Hydrogen bonds, electrostatic interactions (phosphate repulsion stabilizes the helix interior), base stacking

Major and minor grooves in DNA

Binding sites for proteins or drugs

Tertiary structure of DNA

Includes supercoiling and nucleosome organization

Nucleosome

Eukaryotic DNA wrapped around histone octamers

B-DNA

Principal form of DNA

A-DNA

11 base pairs per turn; base pairs not perpendicular to helix

Z-DNA

Left-handed helix

Erwin Chargaff

Showed DNA contains purines and pyrimidines in equal amounts; strands are antiparallel

Rosalind Franklin

X-ray diffraction data revealed double-stranded helix with inward-facing bases

James Watson

Noted A-T and G-C base pairs have similar lengths

Francis Crick

Proposed double helix structure of DNA

messenger RNA (mRNA)

RNA transcript used to make protein

Prokaryotic mRNA

One mRNA can code for multiple polypeptides

Eukaryotic mRNA

Each mRNA codes for one polypeptide and has a more complex structure

hnRNA (heterogeneous nuclear RNA)

Initial eukaryotic mRNA containing both introns and exons

Introns

Non-coding sequences removed from hnRNA during processing

transfer RNA (tRNA)

Transfers specific amino acids to the ribosome during protein synthesis

tRNA structure

73-94 nucleotides

anticodon, acceptor stem, T & D arm

Contains methylated bases

Aminoacyl-tRNA

Active form of tRNA with an amino acid attached

tRNA acceptor stem

CCA-3'-OH forms ester bond with amino acid

tRNA anticodon loop

Binds to mRNA codon via complementary base pairing

tRNA Secondary structure

Cloverleaf, stabilized by intrastrand hydrogen bonds

tRNA loops

Non-hydrogen bonded regions of tRNA structure

ribosomal RNA (rRNA)

Structural and functional component of the ribosome

two-thirds of the ribosome’s mass (the rest is protein).

rRNA structure

Complex secondary structure with intrastrand H-bonds

Can contain pseudouridine and ribothymidine

Prokaryotic ribosome size

70S

Prokaryotic small subunit

30S with 16S rRNA and 21 proteins

Prokaryotic large subunit

50S with 23S, 5S rRNA and 31 proteins

Eukaryotic ribosome size

80S

Eukaryotic small subunit

40S with 18S rRNA and 33 proteins

Eukaryotic large subunit

60S with 28S, 5.8S, 5S rRNA and 49 proteins

snRNA (small nuclear RNA)

Involved in mRNA splicing

Non-coding RNA

Includes introns and regulatory RNAs

Codon

Triplet of RNA bases that corresponds to a specific amino acid

Codon recognition

Recognized by aminoacyl-tRNA

Genetic code

Triplet sequences in mRNA converted to amino acid sequences by the cell

Universality of genetic code

Used by all organisms

Degeneracy of genetic code

Multiple codons may code for the same amino acid

Second genetic code

Mechanism by which aminoacyl-tRNA synthetases attach the correct amino acid to tRNA

DNA replication

Copying of DNA during the S phase of the cell cycle via complementary base pairing

Replication directionality

Bi-directional and semi-discontinuous with 5' to 3' synthesis

Semi-conservative replication

Each new DNA contains one old and one new strand

DNA gyrase/Topoisomerase

Relieves supercoiling ahead of the replication fork

Helicase (DnaB)

Unwinds DNA by breaking hydrogen bonds between base pairs

Single-strand binding proteins (SSBPs)

Stabilize separated DNA strands

Primase (DnaG)

Synthesizes RNA primers with a free 3'-OH

DNA Polymerase III

Main enzyme that extends new DNA strand from RNA primer

Leading strand

Synthesized continuously, sense strand

Lagging strand

Synthesized in fragments called Okazaki fragments

DNA Polymerase I

Replaces RNA primers with DNA

DNA Ligase

Seals nicks between DNA fragments

Beta clamp

Tethers DNA polymerase to DNA for processivity

DNA synthesis mechanism

3'-OH attacks alpha phosphate of incoming dNTP; PPi released

E. coli DNA polymerase III

III = Main enzyme for replication

I, II, V = Involved in DNA repair

Watson & Crick

Predicted that DNA can be copied using each strand as a template

Arthur Kornberg

Discovered DNA polymerase I

Promoter

DNA region where RNA polymerase binds and transcription starts ontains specific recognition sequences (e.g., TATA box in eukaryotes).

TATA box

AT-rich sequence in promoter recognized by RNA polymerase

is easier to unwind

Terminator

DNA sequence where transcription ends

Antisense strand

DNA strand that is transcribed into RNA (template strand)

Sense strand

DNA strand not transcribed; identical to mRNA except T ↔ U

Transcription

Process where DNA is copied into RNA by DNA-dependent RNA polymerase

Reads 3' to 5', synthesizes RNA 5' to 3'

Transcription substrates

rNTPs (ATP, GTP, CTP, UTP) and Mg²⁺

RNA polymerase holoenzyme

Core enzyme (α₂ββ′ω)

σ factor (Directs RNA polymerase to promoter in prokaryotes)

Initiation process(transcription)

RNA polymerase binds promoter

forms RNA polymerase:closed promoter complex

RNA polymerase unwinds ~12 pairs of DNA, forming open promoter complex

RNA polymerase adds NTPs

Initiation site

first NTPs are paired and joined here usually with purine (ATP/GTP)

Elongation site

binds the second incoming NTP

3’-OH of first NTP attacks α-phosphate of second NTP → phosphodiester bond. Releases pyrophosphate (PPi).

Eukaryotic initiation

General transcription factors (GTFs) recruit RNA polymerase II

Elongation (transcription)

RNA polymerase synthesizes RNA 5’ → 3’

After 6–10 nucleotides, σ subunit dissociates forming core polymerase

DNA is unwound ahead and rewound behind (topoisomerases/gyrases relieve supercoiling ahead and behind the bubble.)

Elongation rate

20-50 bases/second

slower in C/G rich regions → 3 H-bonds makes it more stable

Termination (transcription)

Rho factor binds the rut site and moves 5’ → 3’ along the RNA.

RNA polymerase slows or pauses at a termination sequence.

Rho catches up, disrupts the RNA-DNA hybrid → RNA is released.

Rho binding site (rut)

RNA site recognized by Rho factor

Rho factor

ATP-dependent helicase that terminates transcription