Answers to Module 1. An obligate aerobe is an organism that has an absolute requirement of oxygen for growth.

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1 Answers to Module 1 Short Answers 1) What is an obligate aerobe? An obligate aerobe is an organism that has an absolute requirement of oxygen for growth. What about facultative anaerobe? 2) Distinguish between moderate and extreme thermophiles Moderate thermophile optimum growth above 50 C Extreme thermophile optimum growth above 75 C 3) Distinguish between bacteria and Bacteria The term bacteria refers to all prokaryotes (ie. domains Bacteria and Archaea) whereas Bacteria is a specific reference to that domain. 4) What structures are associated with motility in bacteria? Flagella are responsible for bacterial movement. The similarly structured pili and fimbriae are for DNA transfer and attachment respectively. 5) What does a DNA dependent RNA polymerase do? It is an enzyme that synthesizes new RNA strands complementary to a specific DNA template. It is involved in mrna synthesis. 6) Distinguish between bases, nucleosides and nucleotides Base = cyclic, nitrogen containing base Base + deoxyribose = nucleoside Base + deoxyribose + phosphate = nucleotide What is a nucleoside triphosphate? 7) What is the nucleoid? This is the compacted form of the bacterial chromosome. This is achieved by supercoiling and RNA bound folding. 8) What causes translation to terminate and give an example? Stop codons are specific codons recognised by release factors RF1 and RF2 rather than charged trna molecules. This process leads to the termination of translation. Stop codons UAG UAA UGA. 9) What are the 4 groups to which secreted E. coli proteins can be placed? These are the General Secretory Pathways 2 are co-translation translocation processes and the other 2 are chaperone dependent.

2 10) What are Okazaki fragments? Okazaki fragments are discontinuously synthesized DNA fragments found on lagging strands during DNA replication. Medium Answers 1) Distinguish between the RNA polymerase core enzyme and holoenzyme E. coli RNA polymerase - consists of at least 4 polypeptide chains - α ( 2) - β - β - core enzyme (α,β and β subunits) binds randomly and synthesised random lengths of RNA - addition of the σ unit (core enzyme + σ = holoenzyme) causes polymerase to bind at specific sequences 2) Describe two mechanisms of transcription termination Two basic mechanisms: (a) Rho-independent termination: - uses secondary structures forming in the mrna to disrupt transcription - dyad symmetry results in the formation of stem-loop structures - results in pausing of transcription and disrupts the 5 portion of the RNA-DNA hybrid helix - stem-loop also followed by 4-8 uracil bases - causes the 3 -end of the RNA-DNA helix to become highly unstable transcription ceases

3 (b) Rho-dependent termination: - requires the Rho protein - occurs at strong pausing sites - Rho binds to single-stranded mrna - RNA polymerase pauses - Rho translocates along the transcript - when Rho reaches the pause RNA polymerase, it activates with it - Rho + RNA polymerase (+ a helicase) results in the dissociation of the polymerase from the transcription bubble 3) Diagram a Gram-negative cell wall Gram + Gram - 4) Diagram the central dogma (include the types of enzymes used) 5) What is trna and what role does it play in translation?

4 - trna forms the interface between codons encoded in mrna and amino acids to be joined - charged trna has an anticodon at one end (mrna recognition) and a covalently bound amino acid at the other (polypeptide synthesis) Large Answers 1) The E. coli chromosome, when stretched out is 1mm long. How does it fit in a cell less than 5 µm long? 2) Outline the process of replication using a detailed diagram of the replication fork

5 3) Twenty percent of E. coli proteins are located outside the cytoplasm. Describe, using suitable diagrams, the process(es) by which these proteins are secreted. The most common mechanism of periplasmic protein secretion is Sec dependent. - newly synthesized proteins are escorted via SecB to cytoplasmic side of the membrane - energy dependent reaction with SecA initiates partial translocation of the protein through the SecYEG channel - signal sequence remains embedded in the channel with the polypeptide chain in the periplasm until a signal peptidase cleaves it at the appropriate recognition site 4) Describe E. coli and why it is used to study microbial physiology

6 - Domain Bacteria - Family Enterobacteriaceae - First characterized in found in the intestinal tract of many animals - E. coli is a Gram-negative rod - temperature optimum of 37 C (Mesophilic) - ph optimum of 7 (Neutrophilic) - doubling time of 40 minutes in minimal media - 20 minutes in rich media - although haploid - can reproduce sexually (F-factor) - supports the survival of a wide variety of plasmids and viruses - complete genome sequence of E. coli K-12 (strain MG1655) is known The E. coli Genome - genome size is > 4.5 megabases - complete sequence is known theoretical open reading frames - function not assigned to all Suitable model organism because: - widely studied and well characterized - easy and quick to grow - capable of sexual reproduction - supports a wide variety of well characterized plasmids and viruses, many of which have been adapted to molecular biology 5) Describe the process of transcription in detail There are 3 stages in the transcription process: initiation, elongation and termination.

7 Initiation - RNA polyerase recognizes a specific target site called a promoter - promoters consist of two conserved sequences located ~10 bp and 35 bp upstream (i.e. away from) the start of transcription site (+1) Elongation - elongation occurs within a transcription bubble - translocation proceeds along template DNA in the 3 -> 5 direction - mrna is synthesized 5 ->3 Termination - either Rho dependent or Rho independent mechanism

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