1

options

2

3

Polysaccharides

• Peptidoglycan• Glycogen, starch• Monomeric units: Hexoses or

pentoses• Uridine (cell wall) or adenine

(glycogen) diphosphoglucose• Synthesis: Oxaloacetate –

PEP – glucose• Synthesis of pentoses:

removal of C (CO2)

Pentoses: synthesis of nucleotides

4

Synthesis of glucose

Synthesis of AA (proteins)

• Families of AA: carbon skeletons

• CAC, glycolysis, pentoses

5

Glutamate and aspartatefamilies

Nucleotides

• Multiple pathways• First intermediate in

purines: inosinic acid• First intermediate in

pyrimidines: uridylate

6

Lipids/fats

• Acyl carrier protein (ACP)

• Built in pairs• Start: 3(malonyl) + 2

(acetyl)• Final assembly:

glycerol

6.1 Cell growth and binary fission

• Process of growth: synthesis of building blocks

• Growth results in division• Cells grows to 2x the

size• Septum forms

(constriction)• Both cells receive equal

amount of materials

7

FtsZ (filamentous temperature sensitive)• Protein involved in

coordinating fission (GTP hydrolysis)

• Divisome• Controls membrane and

wall synthesis• DNA replication• Ring formation• Other proteins (Min help

locate the middle of the cell)

• Segregation of DNA• Ring depolymerizes and

inward growth• sealing

Cell shape

• Specific proteins• Homologous to actin (eukaryotic)• MreB – assembles in the inside of

cytoplasmic membrane and provides resistance

• Cocci cells lack MreB

8

6.2 Peptidoglycan and cell division• Coordinated process• Autolysins make holes from the inside of cell wall• New material has to be added and mixed without

bursting the cell• Cut and glue new material process

Bactoprenol• Building unit for pg• Highly hydrophobic• C55 compound• Interacts with enzymes

that insert it in the growing point

9

• Transport across membrane

• Bond to existing M-G

Transpeptidation

• Penicillin binding proteins (PBPs)• Cleavage of D-ala provides energy (no

ATP)

10

The great plate count anomaly

• Difference between count and viable• Count > viable

– Dead cells– Within live cells: fraction of cells have special

requirements for culture

6.13 Oxygen and microbial growth• Other options to the use

of O2 as electron acceptor

• Habitats wit low O2tension

• Aerobes• Microaerophiles low

levels (labile enzymes)• Facultative• Anaerobes

– Aerotolerant– Obligate

11

• Clostridium• Archaea• Anaerobic jar (palladium)• Thioglycolate/resazurin

(reducing agent/indicator)

Toxic forms of O2

• Normal state- Triplet oxygen 3O2

• Toxic (reactive) - singlet oxygen (1O2) produced by photo reactions or peroxidases

• Carotenoids prevent oxidative damage• protection

12

Enzymes that destroy toxic oxygen

• Facultative/aerobes have catalase and SOD

+-

13

Chapter 7Principles of Molecular

Biology

7.1 Macromolecules and genetic information

• Coding of genetic information• Transfer of genetic information• Provides powerful tool to modify

organisms• Functional unit of genetic

information: gene• Function -specify the structure of a

polypeptide• Genes- stored information• Proteins- functional entities• Genes- composed of

Deoxyribonucleic acid• Translated into proteins through

RNA (ribose)- intermediary molecule

• RNA can also be part of the cell machinery

• DNA/RNA/proteins- informational molecules

Information coded by base sequencePurines/pyrimidines-AA

Information flow

intermediate

codon

14

Gene

• Genetic functional unit:– Codes (sequence) for a product – Mechanics: DNA-RNA-Protein– Product: polypeptide or RNA

• Polypeptide: AA are coded in sets of three nucleotides

• RNA: rRNA, tRNA– Contains: activation and coding regions

• Promoter, start, code, stop

P

Gene A

atg taa

messagemRNA

Mature protein

15

• Transfer of information is unidirectional: nucleic acid – proteins

• Central Dogma of molecular biology• Exceptions – virology: RNA is used as

template and Reverse transcriptionEukaryotes vs prokaryotes genetics

• Differences in organization of genetic info• Presence of a nucleus

• Messages usually polycistronic –more than one gene is present in transcriptional units

• No non-coding regions

16

• Genes contain coding and non- coding regions (nucleus)

• During transcription non-coding regions are processed (excision-mature)

• Translation (cytoplasm)• Monocistronic mostly

Structure• DNA- double strand• Backbone P-S-P-S: 5-3• Antiparallel- head to toe

arrangement

Specific pairing

17

• Head to toe• Wrapped around-double

helix• Major and minor grooves• Major groove-protein

interactions• One helical turn: major +

minor 10 bp-3.4 nm• Atom exposure-protein

interactionSize

• Molecular wt 330/nucleotide

• Number of base pairs

Sequence specific structures• Important because of

influence on secondary structure-interaction with proteins

• DNA Bends-5 or 6 A runs separated by 5/4 bases

• Bends are aided by proteins• Bends are important in gene

regulation• Repeated sequences also

influence secondary structure-two fold symmetry

• Important in transfer RNA and ribosomal RNA

18

• Stretches of single strands

• When they are complimentary-formation of a circle (sticky ends)

• Circular chromosomes

Single strand

Hairpin

• Similar to stem and loop, but with no loop• Generated at the end, single strand,

presence of inverted repeat

19

Effect of temperature• Under room

temperature and salt conditions kept as double stranded molecule

• Increase in temp will break h-bonds: separation of the strands

• Hybridization-rejoining process

Supercoiling• Relaxed molecule-number of turns by

knowing the number of base pairs• Higher order due to size constraint• E. coli 4.6 mb (0.34 nm/b)-1.5 mm: cells

supercoils DNA to be able to pack it

Further twisted

20

• Supercoil DNA torsion: positive/negative

• Negative opposite to that of right handed double helix

• Histones-proteins that hold DNA tightly wound

• Nucleosomes: combination of proteins and DNA

21

• Nonuclesomesintroduce (-) supercoils

• Placed on the double strand regularly

• Aggregates of nucleosomes-chromatin (visible during cell division)

• Topoisomerases: enzymes that supercoil prok. DNA

• DNA gyrase (Topo II): negative supercoils• Antibiotics inhibit Gyrase• Topoisomerase I: removes supercoils by

creating a nick- double strand break- relaxes• Supercoiled domains prevent total relaxation• Supercoiling –packing relaxing- replication

22

• Supercoiling level: Gyrase –topoisomerase I

• Gene expression affected by supercoiling• Reverse gyrase: positive supercoil

(hyperthermophiles-Archea)histone and and nucleosome like structures• Overwhinding of DNA (protection)

23

Genetic elements

• Structures that are replicated• Genome: full complement of genes• Chromosome (s) (bacteria usually have one),

circular (Borrelia-linear: hairpins?)• Eukaryotes: linear, multiple• Telomere and centromere: sequences important

for partition and replication• Presence of introns and exons (variable)• Much more DNA than coding (repetitive)

Other structures contained in cells

• Viruses: DNA/RNA genome, replicate by integrating into host chromosome

• Plasmids: small, separate from chromosome, circular, non-extracellular, no damage to cell, confer other non essential functions

• Mitochondria and chloroplasts: in eukaryotes, machinery for replication

• Transposable elements: pieces that jump from one site to another site in the chromosome, genetic variation, replicate as part of some other DNA molecule

24

7.5 DNA replication template and primers

• Replication required to grow, reproduce

• Accurate transfer of sequence• Replication: opening of double

strand so a new one can be formed• Semiconservative: formation of 2

double strands, each with one old and one new

• Template: strand copied

New strand synthesis

• Grows from 5 to 3 mediated by cleavage of phosphate bonds

• DNA polymerase needs the presence of OH from a previous nucleotide group to grow the new strand

• DNA polymerase needs a ”primer” (short stretch of RNA)

25

Templates and primers (2)

• Beginning of replication• RNA polymerase enzyme- primase• DNA polymerase• Removal of primer

7.6 The replication fork

• Model E. coli - circular chromosome

• Location: origin of replication• 300 bases in length recognized

by proteins• Open double strand and

replication occurs in bidirection• Point of replication: rep. fork