Microbiology and Immunology 2500A/B Lecture Notes - Lecture 3: Rna-Dependent Rna Polymerase, Reverse Transcriptase, Smallpox

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greyhedgehog626

23 Feb 2016

School

Western University

Department

Microbiology and Immunology

Course

Microbiology and Immunology 2500A/B

Professor

Kelly Summers

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Document Summary

Goal is to make mrna, whatever start with will always make mrna. Mrna= +ve strand that"s ready to be translated. +dna strand= equivalent polarity to +rna strand. Rna/dna complements of +strands = -ve strands. Viral genomes must make mrna which must be read by host ribosomes in the 5" to 3" direction. Central dogma of molecular biology: double stranded dna=>transcription=>single stranded. Viruses dont follow the dogma (bcz starting material/genome can be different) The 7 types of genomes(virus can have dna or rna based genome, but not both): Rna: single stranded +rna, ss-rna, double stranded rna, ss+rna w/ dna intermediate. Dna: ss dna, dd dna, gapped dd dna. Rna viruses(more efficient in making proteins) are much more ancient than dna viruses(more complex) How dna based genomes get their message across(how do they function) Double stranded dna : infect host &come in w/ their ds dna =>transcription using dna dependent. Respects the dogma, very complicated and diverse.

Related Questions

Class 6: Viruses (and Cells, continued)

Making connections: viruses and microbes Match the cell biology concept (on page 1) with an important application of that concept (on page 2). Then answer the follow-up questions at the bottom of page 2.

Cell Biology concepts
1. Bacteria that cannot construct new cell wall material become fragile and break down.
2. During influenza infection, the viral enzyme neuraminidase is required to release new viruses from the host cell in which they were made. Once released, the new viruses can infect many additional cells.
3. Thermus aquaticus is a bacterium found in hot springs and can live at temperatures up to ~80Â°C (175Â°F). It has adapted so that its enzymes are most functional at the elevated temperatures, compared with many organismsâ enzymes that function best ~37Â°C (98Â°F).
4. Until the isolation and investigation of retroviruses, scientists thought that RNA could be copied from DNA, but DNA could not be copied from RNA. Research on this class of viruses led to an update to this âCentral Dogmaâ of biology.
5. Some bacteria have adapted, so that the enzymes involved in making cell wall materials have a slightly different shape and can no longer be bound by certain drugs.
6. Rhizobia are bacteria that colonize the roots of certain plants (legumes, including beans and peas). The host plant benefits from the prokaryotesâ ability to âfixâ nitrogen, converting N2 from the atmosphere into a form that is usable by the plant.
7. Many species of bacteria contain the enzyme cellulase. More than 10 billion such bacteria and other symbiotic microbes may be found in a single milliliter of material from inside the digestive system of ruminant mammals.
8. Fungi, including those that cause athleteâs foot, use ergosterol to maintain healthy membranes. Animal cells do not make ergosterol, they use a similar lipid â cholesterol.
9. During retrovirus infection, a DNA copy is made of the virusâs RNA genome. A cut is made in the host cellâs DNA and the DNA copy of the viral genome is pasted into the host chromosome by a viral enzyme called integrase.
10. Prokaryotic cells contain smaller ribosomes (â70Sâ ribosomes) than eukaryotic cells (â80Sâ ribosomes). Both types of ribosomes synthesize proteins, but the macromolecules they use to do it are somewhat different.

2
Applications
_______ Oseltamivir, also called Tamiflu, binds and inhibits the viral protein neuraminidase.
_______ Clotrimazole is a drug that is used to treat certain infections. It interferes with synthesis of ergosterol, a lipid involved in phospholipid membranes.
_______ Penicillin is a drug that interferes with synthesis of peptidoglycans (these are biological molecules that are part protein and part carbohydrate) that are present in certain cell walls.
_______ HIV is often treated with several drugs that inhibit different parts of the viral replicative cycle. Raltegravir is an example of a drug that interferes with the integration of viral genes into the host cellâs chromosome.
_______ Cows, like other vertebrates, lack the ability to breakdown cellulose. Yet they are able to live on a diet of grass and other vegetation high in cellulose.
_______ Enzymes isolated from extremophiles have been very useful in laboratory research. For example, Taq polymerase facilitates PCR, a revolutionary technique for production of DNAs of particular sequences, which requires cycling reactions through high temperatures.
_______ Plants and animals require nitrogen to make biological macromolecules, however theyâre unable to convert atmospheric nitrogen (N2) into a biologically useful form.
_______ Azithromycin is a drug that binds to 70S ribosomes and blocks synthesis of any new proteins.
_______ Methicillin-Resistant Staphylococcus aureus (MRSA) can cause life-threatening infections of the skin and bloodstream. One of the reasons MRSA infections are so serious is that they are resistant to treatment by many antibiotics, including methicillin and penicillin.
_______ The enzyme reverse transcriptase, isolated from retroviruses, is an important tool in molecular biology research. Scientists interested in learning what genes are turned on in particular cells use it to make DNA copies of RNAs they are studying (The DNA copies are useful because they are more stable).

When you have completed the matching activity, answer these follow-up questions:
â¢ What types of infections can be treated with the following drugs?
o Penicillin
o Clotrimazole
o Azithromycin
o Tamiflu
o Raltegravir
â¢ Why are these drugs (above) safe for you to take? That is, why donât they kill your cells?
â¢ Cancer cells are your own human cells that have become deregulated so that they divide and pile up in an uncontrolled way. Many chemotherapy drugs treat cancer by interfering with cell division and other cell processes.
o Why are there so many more side effects for individuals undergoing chemotherapy than for individuals taking antibiotics?

Please help on these. Thank you!

Question 1

Which of the following is NOT a channel in the RNA polymeraseenzyme?

		RNA exit channel
		DNA entry channel
		DNA exit channel
		Nucleotide exit channel

Question 2

A promoter region upstream of a bacterial gene typically has thesequence TTGACA in the -35 position and TATAAT in the -10 position.If there was a 5 bp insertion at the -40 position, such thateverything downstream was shifted (ie: the "-35 element" is now atthe -30 from the transcription start site), which of the followingwill occur?

		Sigma protein will not bind and so no transcription willoccur.
		RNA polymerase will not bind and so no transcription willoccur.
		Sigma protein will bind to its target sequences and sotranscription will start 5bp downstream from the originaltranscription start site.
		Sigma protein will bind as usual and transcription will startappropriately at the transcription start site.

Question 3

A person has a mutation that prevents TFIIH from being produced.Which of the following outcomes will occur?

		No transcription because TFIID will not be able to bind.
		No transcription because the DNA strand will not be opened.
		Transcription will occur, but it will not be in the correctposition.
		Transcription will occur as usual.

Question 4

There are 4 rRNAs produced to make both subunits of theribosome. How many transcripts are required to produce these 4rRNAs?

		1
		2
		3
		4

Question 5

True or False: the 5' 7m-Gppp cap is attached to the start codonof the mRNA.

		True.
		False, the cap is attached to the 5'-untranslated region.
		False - the cap is attached to the 3'-untranslated region.
		False, the cap is attached to the polyA tail.

Question 6

During the splicing mechanism, which of the following joins twoexons together?

		A 2'OH attacks a 5' phosphate
		A 5' phosphate attacks a 2'OH
		A 3'OH attacks a 5' phosphate
		A 5' phosphate attacks a 3'OH Question 7 After their experiment in Neurospora, Beadle and Tatum concludedthat each single genes code for single enzymes. Do you agree withtheir statement, why or why not?

burgundyhedgehog579

ANSWER THE FOLLOWING QUESTIONS(COULD BE MORE THAN ONEANSWER)

19) Describe the roles of M13 genes and their proteins:

a) Genes 1, 4 and 11 produces proteins required for assemblingthe the phage.

b)Genes 3.6,7,8 and 9 produces capsid proteins.

c)Gene 2 nicks the circular phage ssDNA to make it doublestranded by DNA polymerase

d)Gene 5 produces single strand binding proteins and gene 10protein makes M13 SSDNA circular.

e)Protein five bound single stranded circular M13 moved to theE. coli membranne and is released out coated will proteins 3, 6, 7,8 and 9 .

2) What is a phagemid and how does it work?

a)it is a recombinant DNA with a filamentous phage genome and aplasmid genome.

b)it carries two origins of replication: eg., oir f1 and oriColE1

c)May carry a gene for selection: e.g. Ampr gene.

d)May carry a marker gene; e.g, lacZ gene with promoter andregulator

e)May contain MCS, as is pUC18.

3)For storing functional gene for future applications you willneed the following:

a)A vector DNA

b) A cloning site in the vector matching with the ends of a geneto be cloned

c)A host organism capable to hold the recombined vector.

d)A marker gene present in the recombinant vector

e)A functional gene of interest.

24)identify which of the following is true for thebacteriophage lambda?

a)It has two major body parts, the head and the tail.

b)The genome is approximately 49 thousand bases long.

c) It has a linear dsDNA with single stranded 12 bases long costerminals at both

d) The single stranded lambda DNA cos terminals appear to be asfollows: (a) GGGCGGCGACCT and (b)CCCGCCGCTGGA

e)it can accept 7-10 kb long foreign DNA replacing itsnon-essential b2 region.

29) For developing a recombinant lambda DNA with a gene ofinterest and expressing the gene in a host you have to take thefollowing steps:

a)Remove the b2 region from lambda DNA using a restrictionenzyme

b)digest the gene of interest, outside its stop and startcodons, with the same restriction enzyme.

c)Recombine the lambda DNA digest with the digested gene ofinterest carrying complementary overhangs with ligase.

d)Form lambda particles by adding head proteins, tail proteinsand packaging enzynes to the recombined lambda DNA.

e)Add the packaged lambda particles to Agrobacetrium tumefaciensand incubate them in culture.

f)Add the packaged lambda particles to laboratory strains ofEscherichia coli and incubate them in culture.

32)The amino acids coded by our genes differ by thefollowing:

a)Polarity

b) Acidity

c)Basicity

d)Aroma

e)Hydrophobicity

38)What is an M13 phage and what are its specialty?

a)They are rod shaped Escherichia coli bacteriophage.

b)ts genome is made of a single stranded circular DNA.

c)Its DNA is 6400 nucloetides long coding for elevenproteins.

d)After infecting a bacterial cell it starts replicating in 10minutes producing 1,000 phages in an hour

e) soon its kills the host Escherichia coli and gets out to theenvironment.

40)You have a gene with BamH cut sites out side its start andthe stop codons. You can use the following procedures to prepare aclone of the gene in pBR322 and select the transformed E.Coli cellscarrying pBR322, except:

a) Digest both the gene and the plasmid pR322 with BamH1

b) Ligate the fragments using T4 DNA ligase.

c)Transform E coli with the reconstructed plasmid

d)Select the cells transformed cells grow in Lb-agar-ampicilinplates

e)Select the cells transformed cells grow inLB-agar-tetracycline plates

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Microbiology and Immunology 2500A/B Lecture Notes - Lecture 3: Rna-Dependent Rna Polymerase, Reverse Transcriptase, Smallpox

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