Prokaryotic chromosome structure and Chromatin

Prokaryotic chromosome structure and Chromatin

Objectives

At the end of this lecture, student will be able to describe the following

• Chromosome structure of prokaryotes in general

• Chromosome structure of E. coli

• Chromatin structure

Content

• Prokaryotic Chromosome structure

• Chromatin structure

Prokaryotic chromosome structure

• Prokaryotic genome vary in size

• 0.16 Mb of endosymbiont (Carsonella ruddi) to 13 Mb of soil bacterium (Sorangium cellulosum)

• Chromosome with single circular DNA molecule

• 4.6 Mb of E. coli chromosome

• DNA is packed in a region of cell

• Unbounded nucleiod

• Has very high DNA concentration, along with certain proteins

• Has single or multiple copies of additional small circular DNA molecule – Plasmids

• Vibrio cholerae –1 Mb circle of DNA + 3 Mb chromosome (Second chromosome)

• Agrobacterium tumefaciens have 2.9 Mb circle + 2.1 Mb linear DNA +0.5 Mb + 0.3Mb circular plasmid

• Ti plasmid used as cloning vector

DNA domains

• Consists of 50-100 domains or loops

• Ends are constrained by binding to structure made of proteins attached to cell membrane

• Loops – 50-100 Kb in size

• Loops may spool through the sites of polymerase or other enzymic action at the base of loops

Chromosome structure

Super coiling of the genome

• E. Coli chromosome is negatively supercoiled

• Individual domains may be supercoiled independently

• Some domains may not be supercoiled

• DNA has become broken in one strand

• Other domains clearly do contain supercoils

• Attachment of the DNA to the protein

• Membrane scaffold act as a barrier to rotation of the DNA

• Domains may be topologically independent

DNA binding protein

• Abundant protein -HU, a small basic (positively charged) protein

• Binds DNA nonspecifically by the wrapping of the DNA around the protein

• H-NS (protein H1)- a monomeric neutral protein

• binds DNA nonspecifically in terms of sequence

• Also known as histone-like proteins

• Compacting the DNA - essential for the packaging of the DNA into the nucleoid

• Stabilizes and constrain the supercoiling of the chromosome

• Half of this is constrained as permanent wrapping of DNA around proteins such as HU

• About half the supercoiling is unconstrained

• RNA polymerase and mRNA molecules, site-specific DNA- binding proteins such as integration host factor (IHF), a homolog of HU, which binds to specific DNA sequences and bends DNA through 140°

Chromatin structure (Chromatin)

• Highly organized complex of DNA, nucleoprotein complex

• Makes up eukaryotic chromosome

• Serves to package and organize the chromosomal DNA

• Alters its level of packaging at different stages of cell cycle

Histones

• Major protein components of chromatin

• Most of the protein in eukaryotic chromatin consists of histones

• Five families, or classes of histones-H2A, H2B, H3 and H4: core histones

• Core histones are small proteins, with masses between 10 and 20 kDa

• H1: little larger at around 23 kda

• All histone proteins have a large positive charge

• 20 - 30% of their sequences consist of the basic amino acids, lysine and arginine

• Histones will bind very strongly to the negatively charged DNA in forming chromatin

Nucleosome

• Basic unit of chromatin

• Composed of approximately 146 base pairs of DNA wrapped in 1.8 helical turns around an eight-unit structure-histone protein octamer

• Octamer consists of two copies each of the histones h2a, h2b, H3, and H4

• Space in between individual nucleosomes is referred to as linker DNA

• Linker DNA interacts with the linker histone – H1

• About 142 hydrogen bonds are formed between DNA and the histone core

• Wrapping of DNA is responsible for negative supercoiling in eukaryotic DNA

Role of H1

• A single molecule of H1 stabilises the DNA at the point at which it enter and leaves the nucleosome core

• Organises DNA between nucleosome

• Nucleosome+H1 – Chromatosome

• In some cases H1 is replace by H5

• Binds more tightly

• Associated with DNA which is inactive in transcription

Linker DNA

• Linker DNA between nucleosome varies betweem > 10 & < 100 bp

• Normally around 55bp

• Nucleosomal repeat unit is around 200 bp

• Comprises of globular protein connected by thin strands of DNA

The 30-nm Fiber

• Large structure of organised chromatin

• Also known as solenoid

• Consists of left handed helix of nucleosome

• Approximately 6 nucleosome per helical structure

• Most chromosomal DNA in vivo is packaged into 30-nm fibre

Nucleosome and associated structures

Higher order structure

• Organization of chromatin at the highest level

• Chromosomal DNA is organized into loops of up to 100 kb

• In the form of 30-nm fiber

• Constrained by a protein scaffold, the nuclear matrix

• Overall structure resembles organizational domain of prokaryotic DNA

Summary

• Prokaryotic chromosome consists of a single circular DNA molecule

• It consists of 50-100 domains or loops of DNA domains that may be supercoiled

• DNA binding protein binds the DNA nonspecifically by the wrapping it around the protein

• Chromatin is a highly organized complex of DNA, nucleoprotein complex

• Consists of histones, linker DNA, 30-nm fibre and nucleosome

• The whole of chromatin is organized into higher order structure similar to that of prokaryotic DNA