MCB 150 121-150 Lecture Notes: DNA Organization, Replication, and Transcription

Eukaryotic DNA Organization

• Eukaryotic cells face the challenge of packing approximately 2 meters of DNA into a nucleus with a diameter of only 5-8 μm. This compaction is essential for fitting the genetic material into the limited space available within the nucleus.

  • The DNA must be organized in a way that allows for both compaction and access for processes like replication, transcription, and repair.

Chromatin Organization

• Chromatin, the complex of DNA and proteins, primarily histones, was first described by R. Kornberg in 1974.

  • Chromatin structure influences gene expression and DNA accessibility.

Nucleosome Isolation and Dissociation

• The fundamental repeating unit of chromatin is the nucleosome.

  • Nucleosomes are composed of DNA wrapped around histone proteins.

Histones

• Histones are small, basic proteins that are a key component of chromatin. They facilitate DNA packaging.

  • Histones are positively charged, which helps them bind to the negatively charged DNA.

• There are five major types of histones: H1, H2A, H2B, H3, and H4.

  • Each histone type plays a specific role in chromatin structure and function.

• Histone sequences are highly conserved among species, indicating their critical importance.

  • The high degree of conservation suggests that histones perform essential and universal functions in DNA organization.

• Bacteria do not have histones but possess histone-like proteins.

  • Bacterial histone-like proteins serve a similar function in DNA condensation.

Core Nucleosome

• The core nucleosome consists of two molecules each of histones H2A, H2B, H3, and H4.

  • These eight histone molecules form the histone octamer.

• It contains 146 or 147 base pairs of DNA wrapped around the histone octamer.

  • The DNA wraps around the histone octamer approximately 1.65 times.

• The packing of DNA and histones into nucleosomes results in a chromatin fiber with an approximate diameter of 10 nm.

  • This level of compaction reduces the length of DNA significantly.

Nucleosome Structure

• The nucleosome structure includes two molecules each of H2A, H2B, H3, and H4.

  • These assemble into the histone octamer around which DNA is wrapped.

Chromatosome Structure

• The chromatosome structure comprises a nucleosome plus a single molecule of histone H1.

  • Histone H1 binds to the linker DNA and the nucleosome to further stabilize the chromatin structure.

Summary of 10-nm Chromatin Fibers

• The 10-nm chromatin fiber consists of the nucleosome components (H2A, H2B, H3, H4), histone H1, linker DNA, and non-histone proteins.

  • Non-histone proteins can include transcription factors, DNA repair enzymes, and other regulatory proteins.

10-nm Chromatin Fibers and DNA Shortening

• 10-nm chromatin fibers shorten the length of DNA by approximately six-fold.

  • This level of compaction is crucial for fitting DNA into the nucleus.

• The next level of organization is the 30-nm fiber.

30-nm Chromatin Fibers

• Possible conformations of 30-nm chromatin fibers include the solenoid model and the flexible zigzag model.

  • The solenoid model suggests a regular, helical structure, while the zigzag model proposes a more irregular arrangement.

Levels of DNA Organization

• The organization progresses from the double helix to 30-nm fibers.

  • Further levels of compaction lead to the formation of chromosomes.

Eukaryotic Cell Cycle

• Chromatin condensation varies throughout the eukaryotic cell cycle.

  • During interphase, chromatin is less condensed, while during mitosis, it is highly condensed.

Interphase Chromatin

• During interphase, chromatin exists in loosely condensed form (euchromatin) and highly condensed form (heterochromatin).

  • Euchromatin is transcriptionally active, while heterochromatin is generally silent.

• Euchromatin is distributed throughout the nucleus and is mostly in the form of 10-30 nm structures.

  • This allows for easier access of transcriptional machinery.

• Heterochromatin is found at the periphery of the nucleus and in pockets elsewhere in the nucleus.

  • Its location and condensed state contribute to gene silencing.

Chromatin Condensation During Mitosis

• As a cell enters mitosis, chromatin condenses further, eventually forming familiar chromosomes.

  • This level of condensation ensures proper segregation of chromosomes during cell division.

Mitotic Chromosome Structure

• A typical mitotic chromosome structure includes the centromere, sister chromatids, telomeres, and chromosome arms.

  • The centromere is the point of attachment for spindle fibers, sister chromatids are identical copies of the chromosome, telomeres protect the ends of chromosomes, and chromosome arms contain the genes.

Condensation to Mitotic Chromosomes

• 30-nm fibers condense to mitotic chromosomes through the formation of 300-nm loops and with the help of scaffold proteins, resulting in a 1400-nm chromosome (2x700).

  • Scaffold proteins provide a structural framework for the highly condensed chromosomes.

From Naked DNA to Mitotic Chromosomes

• The process involves multiple levels of compaction to transform naked DNA into mitotic chromosomes.

  • This multi-step process ensures that the DNA is both protected and efficiently organized for cell division.

DNA Replication Summary

• DNA replication is semiconservative, with each daughter double helix consisting of one parental strand and one newly synthesized strand.

  • This ensures genetic continuity from one generation to the next.

• Replication initiates at the origin (ori).

  • The origin is a specific sequence on the DNA where replication begins.

• Synthesis occurs only in the 5' → 3' direction.

  • This is due to the enzymatic activity of DNA polymerase.

• The process requires a single-stranded DNA template, free NTPs and dNTPs, and nucleic acid-synthesizing enzymes.

Additional Requirements for DNA Replication

• DNA replication also requires unwinding enzymes, stabilizing proteins, and glue (ligases).

  • Unwinding enzymes (helicases) separate the DNA strands, stabilizing proteins prevent the strands from re-annealing, and ligases join the Okazaki fragments.

• DNA polymerases require a free 3'-OH group, which is provided by the primer.

  • The primer is typically a short RNA sequence.

• RNA-synthesizing enzymes require a free 3'-OH for synthesis but can hybridize a nucleotide to a nucleic acid strand.

Action at the Replication Fork

• The replication fork is the site of active DNA synthesis.

  • It is where the DNA strands are separated, and new strands are synthesized.

Realistic View of the Replication Fork

• A more realistic view of the replication fork includes the various enzymes and proteins involved in the process.

  • This includes DNA polymerase, helicase, primase, and single-stranded binding proteins.

Transcription in Bacteria

• Transcription is the process of making RNA from a DNA template.

Announcements

• Exam 2 sign-up is available from Wednesday to Friday (Mar. 12-14) due to Spring Break.

• Exam 2 content ends with the lecture on Monday, March 10.

• The lecture on Wednesday is not part of Exam 2.

• There is no lecture on Friday the 14th.

• Wednesday afternoon Student Hours will still be held (4:00-5:30 in 124 Burrill Hall).

• Friday, March 14 will have extended zoom hours for drop deadline conversations.

Why Transcription?

• Transcription provides amplification, as most genes exist in 1 or 2 copies, which is very little template for making proteins.

• The central dogma: DNA → RNA → Protein.

Transcription Overview

• Transcription is carried out by a DNA-dependent, RNA-synthesizing enzyme called RNA Polymerase.

• It occurs in 4 basic steps: Promoter Recognition, Initiation, Elongation, and Termination.

E. coli RNA Polymerase

• E. coli RNA Polymerase copies DNA into RNA and consists of 6 subunits (α2ββ'ωσ), together called the Holoenzyme. Without the σ subunit, it is called the Core Enzyme.

• It doesn't transcribe the entire genome into RNA but looks for individual genes (regions of useful information).

• Promoters serve as