Exam 3

DNA: deoxyribonucleic acid
* double-stranded molecule
*involves 4 bases: A,T,C,G
*bases held together in a string by "sugar-phosphate backbone"
*DNA while strands will uncoil at high temp. DNA is not harmed why heat like proteins are
*nothing magic about DNA
*bacteria genetics not different (in basic) from any body else's genetics

I. So there were mice in 1928 Griffith Experiment
The discovery of DNA
*take salene and inject mix of bacteria/salene into mouse (glossy)
*Mouse dies
*take mix inject mouse (rough)
*mouse lives

glossy - dead
rought - lives
glossy killed by heat - lives
glossy/rough - dead

hypo 1: proteins
support for proteins made up of amino acids (20 amino acids) and lining things are complex so prolly a complex kind of chemical forms our instructions.

Moral of Griffith Experiment:
Molecule of inheritance/genetic material/instructions for critter NOT destroyed by heat, and thus NOT protein.
Laid ground work for discovery of DNA.
Some kinds of genetic exchange occured between dead glossy bacteria and live rough bacteria

Mouse gets injected with: Dead glossy bact. & DNase - Live rough bact. = Mouse Lives!!

II. Properties of DNA
a) supercoiled - all wrapped on itself, so we can fit it all in cell
*DNA gyrase
*Topiosomerase IV
both of these have bacteria enzyme that contained DNA

b) Antiparallel double strands
*2 strands pointed in different directions

What is DNA? Bases attached to a sugar phosphate backbone
5' - 3'
3' - 5'

DNA can only be replicated in 5' - 3'

c) Complimentary base-pairing
A = T and C =_ G can form hydrogen bonds

Semiconservative replication
per copy, only 1/2 is new

III. Steps of DNA replication
a) on bacteriums chromosome, DNA copying begins @ OriC site: lots of A&T, NO C&G.
*Uncoil some DNA: DNA gyrase
*Unzip strands

Dna A (an enzyme)protein binds to bases at OriC on bacteria chromosome
Helicase (another enzyme) binds to Dna A - OriC complex
Helicase starts unwinding DNA at OriC, starts "unzipping" DNA strand
Single - strand binding protein attaches to unzipped DNA at "replication bubble"
DNA polymerase attaches to DNA Primer
small swatch of DNA, complementary to few bases that help start DNA replication
Primer's degrade quickly

Note: DNA polymerase can only move in a 5' - 3' direction
Note: Helicase moves ahead of DNA polymerase, unwinding and unzipping DNA as it goes

One strand synthesized continuously (one long strand with no breaks)::: Leading strand
Other strand synthesized discontinuously (stops, goes, stops, goes)::: Lagging strand

Okazaki fragments - patched together by DNA ligase

DNA polymerase falls off chromosome at terminus site

B/c bacterial chromosomes are circular after replication chromosomes are interlocked.
So, topoisomerase IV cuts both strands of 1 chromosome, de-interlocks circles, patches broken chromosome back together.

Last Step:: Methyl groups added to some bases in DNA = methylation

OriC site - start DNA replication
DnaA - Helicase (both enzymes)
"replication bubble"
SSB protein covers strands of DNA
DNA polymerase, 5'-3' brings in new matching bases, synthesizes new sugar-phosphate backbone
Okazaki fragment patched together with DNA ligase
DNA polymerase reaches terminus site, falls off
Topoisomerase IV, de-interlocks, circular chromosomes
Last step: New DNA starnd has methyl groups added to A & T's

E. coli replicates DNA at a rate of 1000 new bases/second
1 mutant if 10 billion were copied

IV. Repair to errors in DNA replication
a) spontaneous mutation rate about 1 mutant base pair per 10 billion base pair copied
Mutation: change in DNA sequence
effect of mutation usually neutral but can be good or bad

b) Genome sizes:
Mycoplasma genitolum - 600,000 base pairs (bps) in chromosome
T. Pallidum - 1 million bps
S. aureus - 3 million bps
E. coli - 5 million bps
B. anthracis - 6 million bps

*Viruses have smaller genomes than M. genitalum
*Mitochondria also has very small genomes (-size of viral genome)
Stuff thatrelies on a host has a maller genome.
Example: pseudomonas aerugonas lives in soil therefore has a larger genome than v.fisheri which lives inside of the Hawaiin bobtail squid.

T. pallidum - must live in host cell
S. aureus - live on people's skin, hard to live in diff. environments
E. coli - live in stomach, food, live in people and in different environments
B. anthracis - lives in soil

*More reliant on other organism, more reduction in genome
ex: viruses use host enzymes to copy viral DNA, so don't need to have own genes for replication enzymes

I. What are viruses?

II. How humans protect ourselves against viruses
Immune defenses
acquired immunity:
exposed to virus - makes antibodies - if re-exposed, won't get sick.
Very few antiviral drugs

Get an acquired immune response, but don't get illness

Disease eradication
If disease caused by microbe that relies on host to reproduce, if you can help microbe from spreading for 1 cycle, you can wipe it out forever.

Oct 12
b) Mismatch repair system
*similar system in bacteria, eukaryotes
How it works!
i)find mismatched bases
ii) Unmethylated strand = one with error (b/c its newer)
*use endouclease = enzyme that cuts 1 strand of (ds) DNA, eats swatch of (ss) DNA
*then, DNA polymerase synthesizes new swatch
*then DNA ligase patches together fragments

V. Protection of the bacterial chromosome from degradation
a) methylation of DNA
advantage: be able to tell own DNA from foreign DNA. (by which we mean, own cell's DNA vs. viral DNA)
b) restriction enzymes
-cut DNA into fragments at specific sequences (linear DNA especially affected by restriction enzyme)
ex: GATA

Nov. 3

I. Transcription: DNA - > RNA

  • Gene expression

- why do genes - proteins at some times, but not others?
Ex: why doesn't V. fisheri glow always?
- whether particular genes are transcribed and translated at onetime vs. another?

3' - 5' opposite from DNA polymerase
Transcription : not entire genome transcribed all the time, only particular genes.
- a sequence of DNA that codes for one protein
- change in gene expression can be caused by change in what sigma factors bacteria make in different conditions

1. Initiation of transcription:
normally, DNA is supercoiled
-transcriptino makes (ss) RNA copy of DNA gene to do that strand of DNA must be uncoiled, unzip
a) RNA polymerase: sigma factors
*RNA polymerase enzyme that copies DNA - RNA subunits unwinds and unzips DNA strands
Sigma Factors
*Differential versions of subunits that initially binds to DNA
*Subunit that initially binds to DNA recognizes particular sequence of bases in DNA
b) Promoter sequence on DNA
*sequence of DNA bases RNA, polymerase sigma subunit initially binds to, SORNA polymerase starts unwinding DNA
c)Rifampin binds bacterial, but not eukaryotic or archaeal, RNA polymerase
*gives us a target for antibiotics

2. Elongation:
RNA polymerase reaches gene beginning, starts bringing in RNA bases complementary to (ss) DNA sequence hooks bases into chain with sugar-phosphate backbone -> ribose and phosphate

3. Termination
*at the end of DNA gene, hairpin loop forms

  • RNA polymerase can't pass hairpin loop, falls off of DNA, soon releases RNA polymerase

II. Kinds of RNA
a) mRNA - messenger RNA
RNA template for protein
codon: 3-bases long set of RNA bases
that codes for a particular amino acid
mRNA is short lived: if not actively being used as template (translated), it dissolves in about 30 seconds
b) tRNA - transfer RNA
clover leaf shaped
charged tRNA has correct amino acids for mRNA complementary codon attached to side opposite anticodon loop
long lived
c) rRNA - ribosomal RNA
main component of ribosomes (80% of ribosome by weight)
Ribosome translater mRNA to protein
long lived

III. Overview of Translation:

  • mRNA has 3-base long codons that are complementary to anticodons on tRNA's

1. 20 amino acids
3 base/codon and 4 bases = 43 = 64 possible codons

c) have mRNA (copy of DNA gene)
*small subunit of ribosome attaches to Shine-Dalgarno Sequence on mRNA, which is upstream of where codons for amino acids to make protein begin - then large subunit of ribosome attaches to small subunit/mRNA
d) at start codon (AUG) translation begins
e) ribosome will bring in anticodons that are complimentary to mRNA codons
amino acids on backs of tRNA will be linked into chain of amino acids
g) stop codons: don't have complementary tRNA instead, stop codon bind's with protein, RF1 (release factor) or RF2 (enzymes that detach last tRNA from its amino acid)
h) polyribosomes: lots of ribosomes translate an mRNA at the same time

Missence: just one base in DNA sequence is changed- change to amino acid sequence
Nonsence: just one base in DNA sequence is changed- stop codon in mRNA, thus short protein
Silent: change to DNA, and thus to mRNA, but no change in amino acid sequence
Frameshift mutation: change in DNA sequence such that, in mRNA, 1-2 bases added/deleted from "reading frame" of mRNA - different amino acid sequence

I. Transformation - DNA donor dead, and may not be a closer relative of recipient.
a) Artificial transformation:
uses artificial plasmids (circle of DNA w/ genes)
: plasmids that's had new genes w/ genes, inserted into it, by humans.
*expose culture of bacteria (E. coli) to plasmids, heat or electric shock bacteria, some will take in plasmids.
ex: insulin

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