Test 4 Concepts

Ch 16.3 DNA packaging & 16.4 The Nucleus

What is the structure of prokaryotic genomes

• Most prokaryotic cells have a single circular DNA molecule with an average size of 20 kilobase pairs

• Nucleoid = heavily compacted genetic material

• The bacterial chromosome (nucleoid) is negatively supercoiled, extensively folded, and complexed to a few proteins

  • Only one chromosome

What is the structure of eukaryotic genomes. What is the size of the human genome

• The human genome contains 3.2 x 10⁹ base pairs in 23 different molecules - 2 copies of each = 46 chromosomes

  • Total length: ~3.5 meters

  • Cell: 30 – 50 μm diameter

  • Nucleus: ~10 μm diameter

• DNA compacted and wrapped around histone proteins to form chromatin

What are the types of histone proteins and what histones form a new structure

• 5 different histone proteins

  • H1, H2A, H2B, H3, H4

• 2 copies each of H2A, H2B, H3, H4 form the histone octamer

• DNA wraps twice around a histone octamer to produce a nucleosome core particle appearing as beads on a string

What makes up the nucleosome

• Core particles (DNA wrapped around histones) are separated by >50 bp = linker DNA

• H2A, H2B, H3, AND H4 make up octamer

• DNA wraps around the octamer

• H1 rests over wrapped DNA and octamer

• Total nucleosome length is ~200 bp

How are nucleosome fragments separated in agarose gel and what does this tell us about enzyme activity w/ DNA

• DNA fragments can be separated in agarose gel based on their size

• DNA banding pattern in roughly 200 base pair increments

  • Shows that nuclease only has access to the linker DNA

  • Enzyme cannot access DNA wrapped around histone

  • Short term nuclease digestion causes the banding pattern

  • Referred to as a DNA ladder

How is chromatid organized in the nucleus

• Decondensed = euchromatin

  • Available for transcription

• Condensed = heterochromatin

  • Not available for transcription

• Nucleosome → 30-nm chromatin → euchromatin → heterochromatin → chromosome

What is the difference between euchromatin and heterochromatin

• Decondensed chromatin = euchromatin

  • Available for transcription

• Condensed chromatin = heterochromatin

  • Not available for transcription; too compact

  • RNA polymerase and transcription factors cannot access chromatin

• Some chromatin is in a euchromatin state while others are in a heterochromatin state

• Chromatin can change states

What are the different histone modifications and how do they affect transcription

• Chromatin ‘packing’ can be regulated by modifying specific lysine residues by:

• Methylation

  • Histone methyltransferase = add methyl groups

  • Histone demethylase - remove methyl groups

  • Leads to tighter packing → less transcription

• Acetylation

  • Histone acetyltransferase = add acetyl groups

  • Histone deacetylase = remove acetyl groups

  • Leads to looser packing → more transcription

• Other chromatin remodeling proteins can reposition nucleosomes to make DNA more accessible

What is the structure of the nucleus

• The nucleus has a double membrane

  • The outer membrane is continuous w/ the rough ER

• Dense nucleolus

  • A biomolecular condensate composed of intrinsically disordered proteins

  • Site where ribosomal RNA fragments are synthesized

• The inside of the nuclear membrane is lined by proteins called nuclear lamina

  • Nuclear lamina is made up of intermediate filament proteins (nuclear lamins)

• Nuclear membrane has nuclear pores

  • Allows transport of molecules into and out of nucleus

What do nuclear pores do

• Allows movement of molecules in and out of the nucleus occurs through nuclear pores

• 3000-4000 per nucleus

How does nuclear transport work for molecules of different sizes

• Molecules < 40 kiloDaltons may diffuse through pores freely

• Most molecules that must cross the membrane are larger than 40 kDa

  • Like histones and RNA Polymerases

• Most are ‘actively’ transported across the membrane (through the pores)

What is the structure of the nuclear pore. What allows large proteins to travel through nuclear pores

• The proteins that line the pore are called nucleoporins

  • Block the pore

  • Portions of nucleoporins extend into the pore and have many FG repeats (phenylalanine and glycine)

    • FG repeats are common in some intrinsically disordered proteins

    • Intrinsically disordered regions allow smaller molecules in and keep larger molecules out

• Proteins larger than ~40kD cannot navigate through this flexible barrier

• Proteins that are allowed to pass through the nuclear pore possess a nuclear localization signal (NLS)

  • A series of lysine and or arginine residues

  • Bound by the protein Importin which is able to navigate through the FG repeats and transport the cargo into the nucleus

What is the nuclear localization signal (NLS)

• A series of lysine and or arginine residues that can bind to importin and pass through FG repeats into the nucleus

• Site directed mutagenesis to alter the NLS in the large T antigen protein
  expressed by the SV40 virus prevents the protein from entering the nucleus

  • Lys → Thr, basic to polar AA

  • T-antigens found all throughout cell but not the nucleus; it can’t get in w/o NLS and importin

• The bright spots show nucleus; dark spots are the nucleolus

What is the process of nuclear import

• Importin binds cargo protein w/ NLS and together they move into the nucleus

• In the nucleus, importin binds Ran-GTP and then releases the cargo protein

• Importin and Ran-GTP (bound to each other) move out of the nucleus

• In the cytosol, Ran-GTP encounters its GAP, hydrolyzes its GTP to become Ran-GDP, and then releases importin

• Ran-GDP diffuses back into the nucleus, encounters its GEF, and then releases GDP and binds a new GTP

How what is the process of nuclear export

• Most mRNA diffuses from the nucleus into the cytosol without exportin or Ran

• Export of other RNA (rRNA, miRNA) from the nucleus requires proteins in the nucleus that possess a nuclear export signal (NES) to bind to the RNA

• The protein exportin binds to the proteins with a NES and also binds to Ran-GTP and together this complex moves out of the nucleus

• In the cytosol the Ran-GTP encounters its GAP, hydrolyzes its GTP to become Ran-GDP, and then releases the exportin and its cargo protein/RNA in the cytosol

• The exportin and the Ran-GDP independently diffuse back into the nucleus where the Ran-GDP encounters its GEF, and then releases GDP and binds a new GTP

How does secondary active transport work

• The location of the GAP (cytosol) and the GEF (nucleus) produce concentration gradients of Ran-GDP and Ran-GTP across the nuclear membrane

  • Ran-GDP is high in the cytosol and low in the nucleus, so it diffuses freely from the cytosol into the nucleus

  • Ran-GTP is high in the nucleus and low in the cytosol, so it diffuses freely from the nucleus into the cytosol

    • Ran always moves down its concentration gradient

  • The gradient of Ran-GTP provides the energy to cotransport exportin + cargo protein/RNA from the nucleus into the cytosol against their concentration gradient

  • The gradient of Ran-GTP provides the energy to cotransport importin out of the nucleus and create a concentration gradient of importin that is high in the cytosol and low in the nucleus

  • This gradient of importin provides the energy to cotransport cargo proteins from the cytosol into the nucleus against their concentration gradient

What is the Central Dogma of Molecular Biology

• A DNA sequence dictates an RNA sequence

• The RNA sequence then dictates an amino acid sequence (a protein)

What is DNA

• The encoded hereditary information

• Functional unit = gene

  • The sequence of DNA that directs the synthesis of a polypeptide

What are the roles of RNA

• RNA transcribed from DNA is translated into proteins

• mRNA important in synthesis of proteins

• rRNA important in synthesis of ribosomes

• tRNA important in transport of RNA molecules to the ribosome

What is the difference between prokaryotic and eukaryotic transcription

• In prokaryotes, transcription and translation can happen simultaneously

  • Bc of lack of nucleus

• In eukaryotes, transcription happens in the nucleus and translation happens in the cytosol

What is coupled transcription and translation in prokaryotes

• Transcription and Translation can occur at the same time

• A given strand of mRNA can be translated multiple times simultaneously

  • Ribosomes can attach to 5’ end and begin to move 5’ to 3’ along RNA

  • Another RNA can attach and begin translation as well

  • Forms structures called polyribosomes