BIOC220 (10)

Chapter 1.4 Genetic Foundations

The Central Dogma (Principle) of Molecular Biology: The genetic information flows from DNA to RNA to protein.

1. The DNA replicates its information in a process that involves many enzymes: replication.   2. The DNA codes for the production of messenger RNA (mRNA) during transcription in three processes termed initiation, elongation and termination.

3. In eukaryotic cells, the mRNA is processed (essentially by splicing) and migrates from the nucleus to the cytoplasm.

4. mRNA carries coded information to ribosomes that "read" this information for protein synthesis or translation.

Traditional dogma (p.921)

Information in DNA is linear

(one-dimensional).

Extended dogma (p.1021)Linear a.a. sequenceof protein determinesits unique 3-D structure,and dictates if and what supramolecular structure it will be a part of.

1

Lehninger Chapter 8 Nucleotides and Nucleic Acids

[1] Introduction(1) Nucleotides ‒ Roles Subunits of genetic material nucleic acids (DNA and RNA) Main energy currency inmetabolic transactions in cell (mostly ATP) Partially regulate metabolic pathways Mediate many hormomal signals (cyclic AMP, cAMP as second messengers) Components of co-enzymes (NAD+, NADP+, FMN, FAD and coenzyme A) Messenger RNA (mRNA) directs ribosomal protein synthesis (the code) Transfer RNA (tRNA) transports amino acids in cell and delivers them to the ribosome Ribosomal RNA (rRNA) makes up about 2/3 of the ribosome – structural and

functional roles Ribozymes have catalytic properties – function like an enzyme DNA stores the genetic code

(2) Chromosomes

Thousands of genes per cell –Genes are a segment of DNA that produce a biologically active product

2 of each chromosome per cell – one maternal, one paternal

Histones mostly made from Arg and Lys

48-240 million base pairs per chromosome

Animation: http://www.biostudio.com/demo_freeman_dna_coiling.htm

[2] Structures of Nucleotides (D(R)NA Building Blocks)

2

Without the PO3 group,

this would be called a nucleoside.

-D-ribofuranose

* In RNA, the sugar is ribose (OH), in DNA, the sugar is deoxyribose (H).

DNA contains thymidine while RNA contains uracil.*Note: the only difference is the methyl group

[3] NomenclatureA nucleoside is shown in pink, the nucleotide is the entire structure with AT LEAST one phosphate.

3

[4] The Anatomy of DNAA phosphodiester bond/linkage joins one nucleotide to another from the 3 ribose oxygen to a phosphate attached to the 5 oxygen (3-5 phosphodiester bond): a string of bases is read 53

Specific long sequences of A,T, G, Cnucleotides in DNA are the repository of genetic information.

Chemical properties of nucleic acids: Highly negatively charged (pKa of the phosphate group ~0) Has a specific polarity (5- and 3-ends) Always write the sequence from the 5-end to the 3-end (in the 53 direction).

4

pA-C-G-T-AOH, pApCpGpTpA, or pACGTA. Due to the 2-OH, RNA is hydrolyzed rapidly in alkaline conditions, while DNA is stable (why DNA is the chosen molecule for information storage). Nucleic acids show strong UV absorption at 260 nm due to the conjugated bases. Hydrophilic backbone (phosphate and pentose residues) and hydrophobic bases

⇒ base stacking (van der Waals and dipole-dipole interactions)

Bases interact through H-bonding forming specific base pairs (A=T (A=U), GC).

[5] The History Friedrich Miescher (1868) ‒ phosphorous containing, acidic “nuclein” from the cell nuclei. Avery, MacLeod, McCarty (1944) ‒ The first evidence for DNA as the carrier for genetic information.

DNA + proteases mouse dies.

5

DNA + DNAses mouse lives

Hershey, Chase (1952) ‒ 32P (DNA) and 35S (protein) Chargaff’s rules (1940s)

- DNA base compositions differ in species.- Different tissues of the same species have

the same base composition. - DNA base composition does not change over time, etc. - A = T, G = C, and (A + G) = (T + C).

Rosaline, Wilkins (1950s) ‒ X-ray diffraction pattern of DNA. Watson, Crick (1953) ‒ Right handed, antiparallel double helix (B-DNA)

- Double helix - 3.4 Å/base- The hydrophilic phosphodeoxyribose backbones face out - The bases are found on the inside. 10.5 residues per turn (36 Å)

Structural Stabilization?1) Electrostatic interactions: negatively charged phosphate groups are located at the outer surface

where they can be neutralized by: a) histones, b) cations (Mg2+), c) polyamines.2) Base interactions: a) hydrogen bonds between bases (dipole-dipole interactions) b) stacking interaction between the planes of adjacent base pairs (van der Waals forces and

dipole-dipole)

6

The Complementarity of the two strands in the Watson-Crick double helix model suggested a mechanism for the transmission of genetic information.

DNA duplex can exist in different 3-D forms.A-form : a dehydrated formB-form : the Watson-Crick structure, standard form, most stable under physiological conditionsZ-form : left handed A short Z-DNA tracts may play a role in gene regulation.

[6] Replication: duplication of both DNA strands (p. 952, Fig 25-4) What is required?Replication enzyme (DNA polymerase)1. All four dNTPs (dATP, dGTP, dTTP, dCTP)2. Mg2+3. DNA template4. RNA primer (synthesized by primase) with a free 3-OH that can be extended 5. Other proteins …6. Leading strand: 53 continuous strand Lagging strand: 53 Okazaki fragments

Read from 3 to 5 Same direction of replication fork

Joined by a ligase Opposite direction of replication fork

[7] Transcription: The conversion of a DNA sequence to RNA

There are three distinct phases to transcription:① Initiation: RNA polymerase recognizes a promoter site (specific sequence) of DNA upstream

to the gene and locally unwinds the DNA to create a template.② Elongation: The polymerase moves along the gene synthesizing a complementary copy of the

DNA template, but using ribonucleoside triphosphates as precursors

7

③ Termination: When the polymerase encounters a termination sequence it releases the RNA and dissociates from the DNA to end transcription.

[8] The Genetic Code: a set of rules that determines how an mRNA is translated into an amino acid sequence, where the sequence is read as triplets called codons. The genetic code is common for most organisms. Each codon specifies an amino acid, except UAG, UGA and UAA (stop codons – where do I end?). Recall:22nd amino acid: Pyrrolysine “UAG does not always act to signal that a protein is complete, but instead can bereprogrammed to act as the genetic blueprint for the 22nd amino acid, pyrrolysine”

AUG, that codes for Methionine, also serves as an initiation codon (where do I start?)

The Genetic Code is degenerate (>1 codon/amino acid)

Fig 27-7, p. 1038

[9] Translation: the formation of proteins from the mRNA code (read in the 5 to 3 direction) by ribosomes

① Activation of Amino Acids: The amino acid is covalently bound to its corresponding tRNA.② Initiation: The mRNA-ribosome complex is formed and the first aminoacyl-tRNA (initiator

tRNA) binds to the first codon.③ Elongation: The other codons are read sequentially by the ribosome that associates with the

appropriate aminoacyl tRNA (amino acids covalently bound to tRNA) and the polypeptide sequence grows from N- to C-terminus.

④ Termination: When the ribosome encounters the stop codon, it releases the polypeptide and ceases protein synthesis.⑤ Folding and Posttranslational Processing: The protein must fold properly to be active (usually facilitated by chaperonins) and may or may not be modified by enzymes (portions cleaved off or substituents added).

Peptide bond formation

8

Fig 27-24, p. 1059

Interactive Tutorials http://www.vcbio.sci.kun.nl/eng/virtuallessons/cellcycle/trans/http://science.nhmccd.edu/biol/bio1int.htm (full of cool animations. Bookmark this!)

[10] Unusual DNA Structures

Palindrome: A common type of DNA sequence where the base sequences of the two strands exhibit 2-fold rotational symmetry about an axis. Self complementary within each strand and may form hairpin or cruciform structure. May play important roles in gene regulation in vivo.

[11] Nucleic Acid Chemistry(1) DNA Denaturation (Melting) Duplex DNA molecules are denatured (melted) at extreme pHs and at high temp. denaturation (melting)Double-helix DNA strands single strands DNA renaturation (annealing) Melting point (tm) at which half of the strands is denatured depends on: pH, ionic strength (lower I, lower tm), the size, and base composition (high GC contents, high tm).

Initiation sites for DNA replication and transcription are often rich in A=T base pairs.

9

Bubbles: Partially denatured DNA

(2) DNA Hybidization:DNA strands with similar sequences will form partial duplexes or hybrid with each other.

The closer evolutionary relationship between species, the more similar their DNA sequences are, and the more extensively their DNA will hybridize.

This property is used to “fish out” (clone) a similar gene from different species, if the gene sequence from a species is known.(3) Mutations Bases undergo very slow, spontaneous chemical alterations in their structure, resulting in

permanent changes on the genetic information (mutations). Responsible for speciation, evolution, and in individual organism, aging and cancinogenesis.

Deamination of cytosine is most prevalent (~100 events per day in a mammalian cell). Thus formed U in DNA is recognized as foreign, and removed by a repair system. Why DNA contains T, not U (Evolutionary triumph!).

10

Why human DNA hybridized much more extensively with mouse DNA than with yeast DNA?

Depurination by hydrolysis of the N--glycosidic bond. UV irradiation: adjacent TT sequence is most susceptible. skin cancer

(4) DNA Sequencing The Sanger method (dideoxy method) (1977)

Template, the DNA to be sequenced,DNA polymerase, Primer (radiolabeled) for initiation,Dideoxy nycleoside triphosphates (ddNTPs) for termination. Run four separate polymerase reactions with each of ddNTP. Run electrophoresis with the four reaction products. Read (complementary) sequence directly from an autoradiogram of the gel.

11

The Dye Termination method: ddNTPS are labeled with a differently colored fluorescent tag. Sequence is read in a single reaction, not 4 as in the Sanger mtd. Now, up to 600 nucleotide sequences can be read by a single run. Human genome project: Complete sequence of 3.2 billion base pair human DNA was

determined.

12

Chapter 1.5 Evolutionary Foundations

All life is composed of macromolecules made of the same 30-40 molecules! Remarkable similarity of metabolic pathways and gene sequences in organisms

modern organisms came from one primordial cell line. Mutation and natural selection allows evolution.

13

Then, where did these biomolecules (amino acids, carbohydrates, lipids …) come from ?

(1) Pre-biotic Evolution1922 - Aleksandr Oparin - theory on the origin of life - postulated a different atmosphere made of methane, ammonia gases and water vapour,

and devoid of oxygen (reducing environment). lightning or heat from volcanoes caused reactions that would form organic compounds primordial soup - organic compounds concentrated in the seassome would have a greater tendency to associate into larger complexes, and certain

materials would facilitate polymerization (joining together of small subunits)Millions of years past …

spontaneous formation of membranes and catalysts

1953 - Stanley Miller and Harold Urey - tested Oparin's theory Mixed NH3, CH4, H2O and H2

exposed to electrical sparks to emulate lightning and analyzed gas phase: (starting gases, CO, CO2) and water phase (amino acids, hydroxy acids, aldehydes and HCN).

CO2, CO, N2 and H2 in the atmosphere.

have shown the production of hundreds of organic molecules including carboxylic acids, nucleic acids, amino acids and sugars. polymerization reactions are enhanced by

inorganic ions (Cu2+, Ni2+, Zn2+) and other molecules.

sources of energy: heat, Visible and UV light, X-rays, gamma radiation, ultrasound/shock waves, and bombardment with alpha and beta particles RNA was likely the preliminary catalyst (enzyme) and information storage molecule, where protein and DNA originated later.

14

(2) “The RNA World” Hypothesis(4.5 bya) Creation of Earth (3.5 bya) Creation of Life

- See also Fig 1-35 (Landmarks of evolution)and 1-36 (Endosymbiosis).

Q: Why DNA and proteins were eventually chosen?

15

The egg and chicken paradox: Which came first, DNA or protein?Some researchers believe that the predecessor of the current DNA-RNA-protein world was an RNA world, in which RNA encoded both information, like DNA does (genetic information storage), and function, as protein does (catalysis).

Two genes are said to be paralogous if they are derived from a duplication event, but orthologous if they are derived from a speciation event.(http://hpv-web.lanl.gov/stdgen/bacteria/analysis/CHLAMY/orthologdef.html) 

16

Orthologs & Paralogs

Genomics: The systematic study of the complete DNA sequences (GENOME) of organisms. (www.genomicglossaries.com)

Functional genomics: Functional genomics aims to discover the biological function of particular genes and to uncover how sets of genes and their products work together in health and disease. In its broadest definition, functional genomics encompasses many traditional molecular genetic and other biological approaches.