BB 10006 Cell Molecular biology Dr MV

BB 10006: Cell & Molecular biology Dr. MV Hejmadi Dr. JR Beeching (convenor) Prof. RJ Scott Prof. JMW Slack

Dr. Momna Hejmadi (bssmvh@bath. ac. uk) Structure and function of nucleic acids Books (any of these): 2) Biochemistry (2/3 e) by D Voet & J Voet Molecular biology of the cell (4 th ed) by 3) Any biochemistry textbook 1) Alberts et al Key websites 1) 2) http: //www. dnai. org/lesson/go/2166/1994 http: //molvis. sdsc. edu/dna/index. htm

Outline of my lectures Lecture 1. Nucleic acids – an introduction Lecture 2. Properties and functions of nucleic acids Lecture 3. DNA replication Lectures 4 -6. Transcription and translation Access to web lectures at http: //www. bath. ac. uk/bio-sci/hejmadi/teaching%202004 -05. htm

Lecture 1 - Outline How investigators pinpointed DNA as the genetic material The elegant Watson-Crick model of DNA structure Forms of DNA (A, B, Z etc) Types of nucleic acids (DNA and RNA) References: History, structure and forms of DNA http: //www. dnai. org/lesson/go/2166 Voet and Voet – Chapter 28

Timeline 1800’s F Miescher - nucleic acids 1928 F. Griffith - Transforming principle http: //www. dnai. org/lesson/go/2166/1994

Discovery of transforming principle n 1928 – Frederick Griffith – experiments with smooth (S) virulent strain Streptococcus pneumoniae and rough (R) nonvirulent strain

Griffith experiment

n Bacterial transformation demonstrates transfer of genetic material What is this transforming principle?

Timeline 1800’s F Miescher - nucleic acids 1928 F. Griffith - Transforming principle 1944 Avery, Mc. Cleod & Mc. Carty- Transforming principle is DNA http: //www. dnai. org/lesson/go/2166/1994

Avery, Mac. Leod, Mc. Carty Experiment

Timeline 1800’s F Miescher - nucleic acids 1928 F. Griffith - Transforming principle 1944 Avery, Mc. Cleod & Mc. Carty- Transforming principle is DNA 1949 Erwin Chargaff – base ratios http: //www. dnai. org/lesson/go/2166/1994

E. Chargaff’s ratios A=T A+G=C+T C=G % GC constant for given species

Timeline 1800’s F Miescher - nucleic acids 1928 F. Griffith - Transforming principle 1944 Avery, Mc. Cleod & Mc. Carty- Transforming principle is DNA 1949 Erwin Chargaff – base ratios 1952 Hershey-Chase ‘blender’ experiment http: //www. dnai. org/lesson/go/2166/1994

Hershey and Chase experiments n n n 1952 – Alfred Hershey and Martha Chase provide convincing evidence that DNA is genetic material Waring blender experiment using T 2 bacteriophage and bacteria Radioactive labels 32 P for DNA and 35 S for protein

Hershey and Chase experiments

Timeline 1800’s F Miescher - nucleic acids 1928 F. Griffith - Transforming principle 1944 Avery, Mc. Cleod & Mc. Carty- Transforming principle is DNA 1952 Hershey-Chase ‘blender’ experiment 1952 Erwin Chargaff – base ratios 1952 R Franklin & M Wilkins–DNA diffraction pattern 1953 J Watson and F Crick – DNA structure solved http: //www. dnai. org/lesson/go/2166/1994

$X-ray diffraction patterns produced by DNA fibers – Rosalind Franklin and Maurice Wilkins$

X-ray diffraction patterns produced by DNA fibers – Rosalind Franklin and Maurice Wilkins

The Watson-Crick Model: DNA is a double helix n n 1951 – James Watson learns about x-ray diffraction pattern projected by DNA Knowledge of the chemical structure of nucleotides (deoxyribose sugar, phosphate, and nitrogenous base) Erwin Chargaff’s experiments demonstrate that ratio of A and T are 1: 1, and G and C are 1: 1 1953 – James Watson and Francis Crick propose their double helix model of DNA structure

Human genome project Goal: to sequence the entire human nuclear genome Public consortium Headed by F Collins Started in mid 80’s Working draft completed in 2001 Final sequence 2003 Celera Genomics Headed by C Venter Started in mid 90’s Working draft completed in 2001 Nature: Feb 2001 Science: Feb 2001 Human genome = 3. 3 X 109 base pairs Number of genes = 26 – 32, 000 genes

DNA, gene, genome? DNA = nucleic acid Gene = segments of DNA that encode protein Genome = entire nucleic acid component of any organism Nucleic acids: made up of individual nucleotides linked together Protein - polypeptides made up of individual amino acids linked together -

Nucleotides Originally elucidated by Phoebus Levine and Alexander Todd in early 1950’s Made of 3 components 1) 5 carbon sugar (pentose) 2) nitrogenous base 3) phosphate group 1) SUGARS DNA 2’-deoxy-D-ribose RNA 2’-D-ribose)

2) NITROGENOUS BASES planar, aromatic, hetercyclic derivatives of purines/pyrimidines purines pyrimidines adenine guanine Note: Base carbons denoted as 1 etc Sugar carbons denoted as 1’ etc cytosine thymine uracil

nucleotide = phosphate ester monomer of pentose dinucleotide - Dimer Oligonucleotide – short polymer (<10) Polynucleotide – long polymer (>10) Nucleoside = monomer of sugar + base Nucleotide monomer

5’ – 3’ polynucleotide linkages 2) N-glycosidic bonds Links nitrogenous base to C 1’ pentose in beta configuration 1) Phosphodiester bonds 5’ and 3’ links to pentose sugar

5’ end 5’ – 3’ polarity 3’ end

Essential features of B-DNA • Right twisting • Double stranded helix • Anti-parallel • Bases on the inside (Perpendicular to axis) • Uniform diameter (~20 A) • Major and minor groove • Complementary base pairing

n n Structurally, purines (A and G pair best with pyrimidines (T and C) Thus, A pairs with T and G pairs with C, also explaining Chargaff’s ratios

Why DNA evolved as the genetic material but not RNA? Maybe because RNA but not DNA is prone to base-catalysed hydrolysis

B-DNA Biologically dominant Right-handed double helix planes of base pairs are nearly perpendicular to the helix axis passes through the base pairs and hence B-DNA has no internal spaces B-DNA has a wide and deep major groove and a narrow and

DNA conformations B-DNA: n right-handed double helix with a wide and narrow groove. A-DNA n major groove is very deep and the minor groove is quite shallow Z-DNA n consists of dinucleotides, each with different conformations 4 stranded DNA n Telomeric DNA

DNA conformations A DNA both form right-handed double helices B-DNA helix has a larger pitch and hence a smaller width than that of A In B-DNA, the helix axis passes through the base pairs and hence B-DNA has no internal spaces, whereas that of A-DNA has a 6 Angstrom diameter hole along its helical axis. The planes of the base pairs in BDNA are nearly perpendicular to the helix axis, whereas in A-DNA, they are inclined from this. Therefore, B-DNA has a wide and deep major groove and a narrow and deep minor groove, whereas A B DNA

DNA conformations Z DNA B-DNA forms a righthanded double helix in which the repeating unit is a nucleotide, whereas Z-DNA forms a left -handed double helix in which the repeating unit is a dinucleotide. The Z-DNA helix has a larger pitch and is therefore narrower than that of B-DNA has a wide and deep major groove and a narrow and deep minor groove, whereas Z-DNA has a B DNA

Types of RNA q. Messenger RNA (m. RNA): Codes for proteins q. Transfer RNA (t. RNA): Adaptor between m. RNA & amino acids q. Ribosomal RNA (r. RNA): Forms ribosome core for translation q. Heterogenous nuclear RNA (hn RNA) q. Small nuclear RNA (sn RNA): involved in post-transcriptional processing

Genetic material may be DNA Double stranded DNA linear human chromosomes circular Single stranded DNA Prokaryotes Mitochondria Chloroplasts Some viruses (pox viruses) adeno-associated viruses circular Parvovirus

Genetic material may be RNA Double stranded RNA Single stranded RNA Retroviruses like HIV reoviruses

RNA / DNA hybrids e. g. during retroviral replication

What is the base found in RNA but not DNA? ? A) Cytosine B) Uracil C) Thymine D) Adenine E) Guanine

What covalent bonds link nucleic acid monomers? A) Carbon-Carbon double bonds B) Oxygen-Nitrogen Bonds C) Carbon-Nitrogen bonds D) Hydrogen bonds E) Phosphodiester bonds

What sugar is used in in a DNA monomer? A) 3'-deoxyribose B) 5'-deoxyribose C) 2'-deoxyribose D) Glucose

Each deoxyribonucleotide is composed of A) 2'-deoxyribose sugar, Nitrogenous base, 5'hydroxyl B) 3'-deoxyribose sugar, Nitrogenous base, 5'hydroxyl C) 3'-deoxyribose sugar, Nitrogenous base, 5'Phosphate D) Ribose sugar, Nitrogenous base, 5'-hydroxyl E) 2'-deoxyribose sugar, Nitrogenous base, 5'phosphate