Genes and Chromatin

Question 1

What are the consequences of the ER being continuous membrane

Answer 1

1. Can form connections with nuclear pore systems, allowing almost direct transport of newly synthesized proteins targeted for function in the nucleus directly into the nucleus 2. Provide newly synthesized and processed RNA to the rough ER for translation / protein synthesis.

Question 2

How does heterochromatin differ from euchromatin?

Answer 2

An electron micrograph of DNA-protein complexes – chromatin – is structurally dynamic with various degrees of condensation (heterochromatin) vs more open and loosely packed chromatin (euchromatin). Euchromatin occurs near nuclear pores. The euchromatin is loosely packed and is generally involved with actively making RNA to be transported out of the nucleus and into the cytosol or ER for conversion into protein. The newly synthesized proteins are returned to the nucleus through the nuclear pore system to help maintain chromatin structure, engage in replication, damage repair, transcription and RNA processing. All of which we will be covering over the next few sessions.

Question 3

Describe the structure and function of the nucleolus

Answer 3

Above is a cartoon and a pair of electron micrographs depicting a nucleolus structure. The nucleolus is a specialized compartment found in the nucleus. It is usually constitutively active in gene expression pumping out lots of ribosomal RNA and RNAs that code for ribosomal proteins. These products are transported out of the nucleolus and then out of the nucleus into the cytosol, where they undergo final assembly to manufacture functional ribosomes to engage in protein biosynthesis. These ribosomes are either “floating free” in the cytosol or are associated with the endoplasmic reticulum (ER). The consequences of this differential ositioning will be thoroughly discussed in the next few sessions.

Question 4

Four characteristics of the nucleus and their roles

Answer 4

Question 5

Describe the chemical nature of a gene

Answer 5

A unit of DNA which can be expressed as RNA

Question 6

What does Central Dogma state?

Answer 6

The central dogma simply understates multiple text books worth of science, painlessly abbreviated to DNA sequences are transcribed into RNA sequences, and RNA sequences are translated into amino acid sequences. The sequence of amino acids fold up into precise 3D structures called proteins.

Question 7

What are the omes of the central dogma?

Answer 7

Genome, transcriptome, exome, proteome, metabolome

Question 8

Genome

Answer 8

–The DNA in one human cell is approximately 2.15 meters in length (over 6 ft, while the cell is only 50 – 200 microns). Packaging is important!

–There are over 3 billion base pairs in the human genome.

–Genes = DNA sequences coding for expressible materials (RNA) and their directly associated DNA regulatory sequences.

–There are approximately 23,000 coding genes in the human genome.

–Up to 20% of the DNA contains these genes (What is the remaining ~80% of human DNA doing?)

Question 9

Transcriptome

Answer 9

–The RNA component, or transcripts, synthesized from coding sequences of DNA genes.

–This does not include DNA regulatory elements involved with packaging or driving transcription of DNA into RNA.

–Not all RNA transcripts code for proteins, some RNAs are vitally important to cell function all on their own as RNA

–Only 1-2% of intact human DNA actually codes for an expressible protein product.

Question 10

Exome

Answer 10

–The RNA component remaining after processing, editing and splicing together exons from the original primary transcript of RNA derived from DNA.

–These are terminally processed and bio-functional RNA transcripts: mRNA, rRNA, tRNA, miRNA, snRNA , lncRNA…

–There are estimated to be upwards of 100,000 variants of expressed RNA (e.g. splicing variants and chemical modifications, regulatory non-coding RNAs)

–The processed mRNA transcripts contain both protein coding and translational regulatory sequences

Question 11

Proteome

Answer 11

–All of the proteins translated from functional mRNA transcripts in a given cell

–Different cell types express different proteomes based on differential gene expression

–Differential processing and protein modifications can lead to over 500,000 different protein products

Question 12

Build Q

Answer 12

Nucleic acids are complex molecules consisting of either ribose (RNA) or 2-deoxyribose (DNA) sugars and purine or pyrimidine nitrogenous bases. These constituent molecules shown above have a specific numbering system to identify the individual atom centers in each molecule. Purine atom centers are numbered 1 – 9, while pyrimidine atom centers are numbered 1-6. Note that the ribose carbon atom are numbered 1 – 5 with a “prime” designation in order to distinguish ribose carbons from purine and pyrimidine carbon and nitrogen atom centers. This is important so that the modifications to the ring structures can be denoted specifically; such as thymine is actually the common name for 5-methyl uracil. 2’deoxyribose indicates that the #2 carbon atom of ribose is missing its oxygen. There are well over 100 chemical modifications to the nucleic acids crucial to molecular biology. These are designated appropriately as follows for example; 2’-acetylribose, 7-methylguanine, N2-methylguanine or 5-methylcytosine, etc

Question 13

What is the difference between nucleosides and nucleotides?

Answer 13

Assembling ribose and purines and pyrimidines into their bio-functional units involves establishing new nomenclature.

Nucleosides = ribose chemically linked to a nucleic acid base via an N-glycosidic bond in the beta conformation (not alpha).

Nucleotides = nucleosides with a covalently bonded phosphate group. Up to three phosphate groups are common on nucleotides.

Question 14

Build q

Answer 14

Nucleotides are covalently linked together via 5’-3’ phosphodiester bonds. These are very stable for DNA molecules. These are less stable for RNA molecules because the 2’OH group can engage in autocatalytic cleavage of the 3’ phosphodiester linkage. This occurs more rapidly in aqueous solutions that are mildly basic (pH > 8.5). RNA is not stable in aqueous solutions, it has a limited life span, if left unprotected by specific RNA binding proteins. DNA is not effected by basic aqueous solutions, WHY?

SO…. DNA evolved from the RNA world to serve as a stable nucleic acid for storing all life’s genetic information.

It is very much implied that the chemistry of the primary structure is intact (5’ à 3’ sequence), as shown above, throughout the genome. Molecular Biologist mercifully abbreviate the need for drawing out all these structures by simply writing out the sequence of the DNA molecule using the letters ATGC. By convention, the left end is the 5’ end and the right end is the 3’ end of the DNA molecule.

BUT … DNA is a double helix, so, what must be the sequence of the other strand of DNA? Complimentary antiparallel single stranded, ring a bell … anyone?

Question 15

What comprises of the secondary structure of DNA?

Answer 15

Behold – years of scientific work and some basic thievery, comes down to a half page paper in Nature describing a DNA molecule as a symmetrical right handed, anti-parallel, double helix of nucleotides joined together by 5’-3’ phosphodiester bonds, with the nucleic acid bases to the internal axis of the molecule and the phosphoribosyl backbone to the external surface of the axial core. The entire thing is stabilized by a vast number of hydrogen bonds specifically between guanine and cytosine, and adenine and thymidine. The general form of the double helix shows a 10 nucleotide stretch of stacked bases per 3.4nm twist/turn of the helix. The general form of the double helix also displays both a major groove and a minor groove in its structure. In addition to the stabilization by hydrogen bonds between AT and GC, the bases are stacked in very close proximity squeezing the water out of the core of the helix. This increases hydrophobic interaction between the psuedo-aromatic nucleic acid bases allowing for a phenomenon called base-stacking interaction via pi-orbital resonance along the entire length of the double helix … this is very stabilizing to the DNA structure. Finally, the two DNA strands of the double helix must be in an anti-parallel orientation in order for the nucleic acid base pairs to approach each other and form productive hydrogen bonding arrangements. If the strands were parallel, the strands would never be able to form hydrogen bonding interactions due to steric hindrance between the various covalent bonds involved. Simple, right?

Question 16

BQ

Answer 16

DNA is a dynamic molecule. It can change its overall and even local shapes based on environmental conditions. The original crystal structures for DNA were generated in a high salt crystal with organized-bonded water molecules. Fortunately these conditions generated a double stranded DNA (dsDNA) that closely simulated the most common native form of dsDNA - the B-form of the dsDNA alpha helix. There are dozens of various B-forms, closely similar to the Watson-Crick dsDNA. There are also numerous other structures that are substantially different. The two ends of the spectrum for DNA structures are A-form DNA and Z-form DNA as shown above.

The A-form has 11 to 12 base pairs per twist and is compressed. It is not flexible and tends to form over shorter regions of AT rich DNA. Note it does not have definable major or minor grooves as does B-form dsDNA. This is key to how A-form DNA interacts with specific proteins. A-form will not interact with the same proteins that bind to B-form and visa-versa. Since protein-DNA interaction is key for DNA stabilization, and gene expression, this dynamic shifting of DNA between various forms can regulate aspects of gene expression. ( a key concept for epigenetics coming up soon)

At the other end of the spectrum is Z-form DNA. It is a left-handed double helix of 8 to 9 basepairs. This form is stable in limited lengths of dsDNA, that are particularly rich in GC content, especially if the C is modified to 5-methyl-cytosine (5meC). Obviously, proteins that bind to the major groove of a B-form DNA will not be able to access the Z-form DNA what-so-ever. This is commonly associated with a phenomenon called gene-silencing. (more epigenetics to ponder)

NOTE: all these forms are dynamically interchangeable through the B-form of DNA. These changes are controlled by local environments; pH, salt, degree of hydration, local primary structures of DNA (sequence), and various chemical modifications that can and do occur to DNA. Its is a very dynamic process with profound molecular biological effects.

Question 17

What is the purpose of dsDNA supercoiling?

Answer 17

Supercoiling is a complex structural occurrence common in DNA, particularly found in the B-form dsDNA. It can be considered a tertiary structure. It is both flexible and dynamic. Supercoiling is essential to the overall packaging and stabilization of DNA with nucleosomes (more later). One supercoil twist per nucleosome is quite stabilizing for chromatin complexes. DNA has a natural supercoil density that must be maintained in order for functional processes, such as replication, transcription, packaging, protection, storage, etc.

Anyone with an old telephone that actually has a cord – I’m dating myself here – can demonstrate what a supercoil looks like.

My preferred model is a length of Tygon tubing, and we will demonstrate this in class.

Above are electron micrographs of circular DNA in various states of supercoil density. These are common occurrences in mitochondrial, bacterial and plasmid DNAs. Human DNA is linear in the form of 23 pairs of chromosomes, more later. But every cells mitochondria has a supercoiled circular DNA mini-mito-genome.

Question 18

What are DNA denaturing agents and how do they work?

Answer 18

DNA can be denatured. The secondary structure (double stranded DNA - dsDNA) can be eliminated and reduced down to a primary structure (single stranded DNA - ssDNA). The above denaturing agents can breakdown the alpha helix very effectively. Note, that the primary structure stays intact, the phosphodiester bonds are not broken in denaturing dsDNA à ssDNA. This characteristic of DNA is vitally important to several powerful molecular biological techniques in both laboratory research and clinical applications (e.g the polymerase chain reaction – PCR).

Temperature, pH, chemical solvents can all reversibly denature dsDNA to ssDNA. Under these conditions the weak interactions between hydrogen bonding and base-stacking are disrupted. Water rushed in and steals all the possible hydrogen bonding interactions and the two strands fall apart, forced by water. Returning to normal temperature, pH or removal of organic solvents by dialysis, will allow for complementary base pairing sequences to reanneal (renature), squeezing the water out and reforming hydrogen bonds and base stacking interactions. This cyclic process can be done over and over again depending on the process being run. Localized denaturing is also of paramount importance in both DNA replication and transcription …. No minor thing!

Note, chemical modification with a compound like formaldehyde, can cause irreversible denaturing of dsDNA à ssDNA. Formaldehyde will interact and form a covalent bond with the nitrogen at position 2 of a guanine ring structure. This will block any further GàC interaction disrupting the dsDNA permanently. This is used in the lab, but in nature this can lead to DNA damage and potential mutation resulting in numerous health issues. Formaldehyde is a known carcinogen, yet it is frequently used in organic fiber processing: carpets, foam pillows, plywood and fiberboard manufacture. Beware of that new car / new home smell … it can kill you!

Question 19

How do major and minor grooves differ and how does this influence their interactions with protiens?

Answer 19

Interactions of proteins with specific DNA sequences occur through the major groove. This is typical of transcription factors which access specific DNA base-pairing sequences. General interactions of proteins with DNA occur with the overall structural motif of DNA. This occurs primarily through interactions with the minor groove. This is typical of interactions with structure supporting histone proteins where positively charged amino acids in histone proteins (lysine and arginine) interact with the negatively charged DNA phosphoribosyl backbone.

Question 20

BQ

Answer 20

There are 5 different histones. Two each of four histones (H2A, H2B, H3 and H4) come together to form an octamer that is the core of a complex called the nucleosome. Its function is to bind to and stabilize DNA. A DNA helix will wrap approximately 1 ¾ times around a single nucleosome complex. This occurs predominantly through electrostatic interaction between the negatively charged phosphoribosyl backbone of DNA and the positively charged Lysine and Arginine residues on the histone proteins making up the nucleosome octamer. This structure is the primary fundamental unit of chromatin (protein- nucleic acid interactions and structures).

Note: in the diagram above that the histones have random coil free ends that protrude from the core nucleosome particle. These polypeptide domains are involved with nucleosome interactions, and are the target of several chemical modifications that affect the interactions of nucleosomes (packaging). These interactions strongly effect overall chromatin structure and gene expression.

Question 21

What is the purpsoe of H1?

Answer 21

Histone H1 is also referred to as “linker” histone. It functions as a “cap” or connector polypeptide/protein to help associations between nucleosome core particles (the octamer). The complex including the H1 associates with and protects 200 base-pairs of DNA. Nucleosome interactions are highly dynamic and are related to genome stabilization, storage, protection and gene expression. Note: 200bps is the common DNA fragmentation profile seen during apoptosis. What does that suggest to your?

Question 22

What are the higher order of chromatin structures?

Answer 22

Tightly packed nucleosomes can form very stable structures. Packing is organized by H1 interactions and a wealth of chemical modification to various histone “tails”. It is at this level where much of the chromosomal organization of chromatin is aided by non-histone scaffolding proteins.