BB10006: 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): Biochemistry (2/3e) by D Voet & J Voet Molecular biology of the cell (4th ed) by Alberts et al Any biochemistry textbook Key websites 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 F Miescher - nucleic acids 1928 F. Griffith - Transforming principle http://www.dnai.org/lesson/go/2166/1994

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

Griffith experiment Figure 6.3 a

Griffith experiment Figure 6.3 b

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

Timeline F Miescher - nucleic acids 1928 F. Griffith - Transforming principle Avery, McCleod & McCarty- Transforming principle is DNA 1944 http://www.dnai.org/lesson/go/2166/1994

Avery, MacLeod, McCarty Experiment Figure 6.4 a

Avery, MacLeod, McCarty Experiment Figure 6.4 c

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

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

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

Hershey and Chase experiments 1952 – Alfred Hershey and Martha Chase provide convincing evidence that DNA is genetic material Waring blender experiment using T2 bacteriophage and bacteria Radioactive labels 32P for DNA and 35S for protein

Hershey and Chase experiments Figure 6.5 a,b

Hershey and Chase experiments Figure 6.5 c

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

X-ray diffraction patterns produced by DNA fibers – Rosalind Franklin and Maurice Wilkins Figure 6.6

The Watson-Crick Model: DNA is a double helix 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 Public consortium Headed by F Collins 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 Nature: Feb 2001 Celera Genomics Headed by C Venter Started in mid 90’s Working draft completed in 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 RNA 2’-deoxy-D-ribose 2’-D-ribose)

2) NITROGENOUS BASES planar, aromatic, hetercyclic derivatives of purines/pyrimidines adenine cytosine guanine thymine Note: Base carbons denoted as 1 etc Sugar carbons denoted as 1’ etc 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 C1’ pentose in beta configuration 1) Phosphodiester bonds 5’ and 3’ links to pentose sugar

5’ – 3’ polarity 5’ end 3’ end

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

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 Figure 6.9 d

Maybe because RNA but not DNA is prone to base-catalysed hydrolysis 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. 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 deep minor groove

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

both form right-handed double helices DNA conformations B DNA 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 B-DNA 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-DNA has a narrow and deep major groove, but a wide and shallow minor groove.

DNA conformations Z DNA B DNA B-DNA forms a right-handed 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. B-DNA has a wide and deep major groove and a narrow and deep minor groove, whereas Z-DNA has a narrow and deep minor groove but a nonexistent major groove. B DNA

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

Genetic material may be DNA Double stranded DNA Single stranded DNA linear linear human chromosomes adeno-associated viruses circular Prokaryotes Mitochondria Chloroplasts Some viruses (pox viruses) circular Parvoviruses are among the smallest, simplest eukaryotic viruses and were only discovered in the 1960's. They are widespread in nature; human Parvovirus infections were only recognised in the 1980's. Essentially, they fall into two groups, defective viruses which are dependent on helper virus for replication and autonomous, replication-competent viruses. In all, >50 parvoviruses have been identified Parvovirus

Genetic material may be RNA Double stranded RNA Single stranded RNA Parvoviruses are among the smallest, simplest eukaryotic viruses and were only discovered in the 1960's. They are widespread in nature; human Parvovirus infections were only recognised in the 1980's. Essentially, they fall into two groups, defective viruses which are dependent on helper virus for replication and autonomous, replication-competent viruses. In all, >50 parvoviruses have been identified 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