Microbiology Exam 2

Griffith's experiment

an experiment using the heat-killed bacteria in mice to discover that a factor in heat-killed, disease-causing bacteria can "transform" harmless bacteria into ones that can cause disease

used pneumococcus to show that DNA was genetical material

Live S strain

killed mice

Live R strain

did not kill the mice

Heat killed S strain

-denatured by heat

-enzymes (protein) change, substrate change

-no longer deadly

Live R strain and heat killed S strain

mouse dies because S strain DNA was transformed into R-strain

transformation

process in which one strain of bacteria is changed by a gene or genes from another strain of bacteria

Avery-MacLeod-McCarty experiment

demonstrated that DNA is the genetic material because degradation of DNA led to a cessation of bacterial transformation

tested enzymes (DNase, RNase, protease)

DNase - no transformation

Avery-MacLeod-McCarty experiment steps

1. Mix R cells and DNA extract from S cells (treated or untreated)

2. Allow DNA to be taken up by R cells

3. Add antibodies that cause untransformed R cells to aggregate

4. Gently centrifuge to remove aggregated R cells, leaving only S cells

Hershey-Chase Experiment

confirmed that DNA is the genetic material because only radiolabeled DNA could be found in bacteriophage-infected bacteria

35S protein

radioactively labelled with sulfur

all protein stayed on the phage

radioactive protein was in the supernatant

32P protein

radioactively labelled with phosphorus

radioactive DNA was in the pellet where the cells were located - showed that DNA was carrying the virus's genetic information

Blender treatment

sheared off bacteriophage particles absorbed to E.coli cells

Watson and Crick

Developed the double helix model of DNA.

bacterial chromosome replication

1. DNA synthesis begins at the origin of replication

2. Synthesis of DNA proceeds bidirectionally around the bacterial chromosome

3. Two replication forks meet at opposite side of chromosome, ending replication

DNA replication is

semiconservative

origin of replication (oriC)

Site where DNA synthesis

begins proceeds bidirectionally

DnaA protein

initiator protein that binds to oriC

9-bp sequence that is repeated 12 times in oriC

DUE

DNA unwinding element

has lots of AT base pairs

two strands separate here

two strands separate here

Why are AT-rich segments better?

only have two hydrogen bonds

become single stranded more readily than GC-rich regions

IHF

DNA-binding protein (integration host factor)

stimulates initiation

Replication bubble

Segment of a DNA molecule that is unwinding and undergoing replication

ter region

both strands meet and replication terminates

Replication fork

place at which the parental DNA helix is unwound and the two strands are replicated

replisome

12 proteins that form a complex at the replication fork

two replisomes move in either direction away from the origin

Helicase (DnaB)

six membered ring that encircles one DNA strand

unwinds the DNA helix by disrupting the hydrogen bonds

powered by ATP

moves the replisomes

In what direction is DNA synthesized?

5' to 3' direction

dNTPs (deoxyribonucleoside triphosphates)

free deoxyribonucleotides needed for extension through phosphodiester bond

Where does the energy to form the phosphodiester bond come from?

release of the terminal two phosphates as pyrophosphate

breaks the bonds

What substrate requirement does DNA polymerase need?

1. template (parental DNA strand)

2. 3' -OH group from growing nucleic acid chain

primase

short 10 base RNA molecule complementary to the template

primer

a short stretch of RNA with a free 3' end, bound by complementary base pairing to the template strand and elongated with DNA nucleotides

primosome complex

primase and helicase linked together

E.coli DNA polymerases

DNA polymerase I, II, III, IV, V

DNA polymerase III

synthesizes new DNA only in the 5' to 3' direction

proofreading - removes mismatched bases immediately after it has been added

DNA polymerase I

Removes RNA nucleotides of primer from 5' end and replaces them with DNA nucleotides

Clamp

attaches to each polymerase and stabilizes the enzyme on the DNA template

Clamp Loader Complex (CLC)

mediates the polymerase attachments to the primosome

SSB

single stranded binding proteins

coat single-stranded DNA to protect from damage

Topoisomerase (DNA gyrase)

relieve the twist generated by the unwinding of the double helix

break and reseal one or both strands

AHEAD of the replication fork

Pol III holoenzyme

17-subunit E. coli DNA polymerase III complex, responsible for chromosomal replication

Parts of Pol II holoenzyme

1. 3 Pol II subunits

2. 2 sliding clamps

3. clamp-loading complex

4. Other enzymes

leading strand

the new complementary DNA strand synthesized continuously along the template strand toward the replication fork in the mandatory 5' to 3' direction

lagging strand

A discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5' to 3' direction away from the replication fork

Okazaki fragments

Short stretches of polynucleotides produced during discontinuous DNA replication

DNA ligase

enzyme that chemically links DNA fragments together

uses NAD+ or ATP

Where does DNA ligase join the fragments?

forms a phosphodiester bond between 3' -OH of growing strand and 5' phosphate of Okazaki fragment

exonuclease activity

ability of DNA Pol III to move backwards to remove a nucleotide from the end of a DNA strand

removes mismatched base from 3' end of growing strand

endonuclease activity

Cuts a polynucleotide in the middle of a chain

Termination of Replication in E. coli

1. catenated chromosomes

2. dimerized chromosomes

catenanes

interlocked circular molecules

How are catenanes resolved?

Topoisomerase I breaks both strands on one piece, pass another through, and reattach on other side

dimerized chromosome

two chromosomes joined together to form a single chromosome twice as long

How are dimerized chromosomes separated?

XerCD recombinase

catalyzes an intramolecule crossover that separates the two chromosomes

Do archaea replisome have higher homology with bacteria or eukarya?

Eukarya

chromosome replication in archaea

1. multiple replication origins with circular chromosomes

2. recognized by a specific initiator protein - ORC

ORC

DnaA homolog in archaea

XerA

XerCD homology in archaea

brings together termini

Unique DNA polymerase in archaea

DNA polymerase D

3 major types of RNA

mRNA, tRNA, rRNA

Central dogma

DNA-transcription-RNA-translation-protein

template strand

DNA or RNA strand that specifies the base sequence of a new complementary strand of DNA or RNA

sense strand

complementary DNA strand

bacterial structural gene

Pribnow box

Promotor sequence at -10

RNA polymerase binding site

promoter region

binding site for RNA polymerase

not transcribed or translated

strictly to orient RNA polymerase so its a specific distance from the first DNA nucleotide

leader region

The region of an mRNA between the 5' end and the initiation codon for translation of the first polypeptide chain

NOT translated into amino acids

Shine-Dalgarno sequence

(AGGAGG)

initiates prokaryotic translation by interacting with rRNA molecules comprising the 30S ribosome

coding region

part of a gene that contains the coded information for making a polypeptide chain

begins with the template DNA sequence 3'-TAC-5'

remainder specifies the amino acid sequence

trailer region

a nontranslated sequence following the last termination codon

prepare RNA polymerase for release from the template strand

terminator

sequence that signals RNA polymerase to stop transcription

tRNA

transfer RNA

type of RNA that carries amino acids to the ribosome

genes for tRNA consist of

promoter

leader

tRNA coding region

Trailer

spacer in tRNA

used when more than one tRNA is transcribed from the promoter

separates the coding regions

intital transcript must be processed to

remove the noncoding sequences (leader, trailer, spacers)

post-transcriptional modification

removal of noncoding sequences

accomplished by ribonucleases

what do trailer regions and spacers often contain?

tRNA genes

precursor rRNA encodes for both tRNA and rRNA

operon

multiple genes under control of one promoter

polycistronic mRNA

mRNA that has more than one coding region

mutliple start and stop codons

formed when an operon is transcribed

usually occur in bacteria and archaea

monocistronic mRNA

mRNA that codes for a single gene

rare in bacteria/archaea

RNA polymerase

enzyme that links together the growing chain of RNA nucleotides during transcription using a DNA strand as a template

RNA polymerase holoenzyme

bacterial RNA polymerase that scans the DNA for a promoter sequence

sigma factor bound to core enzyme

sigma factor

has no catalytic activity

transcription factor by helping the core enzyme recognize the -35 region

sigma factor 70

bind to the consensus TATAAT at -10 (E. Coli)

sigma factor 54

nitrogen metabolism genes

sigma factor 38

stationary phase and stress response genes

sigma factor 32

heat shock response

sigma factor 28

motility genes

sigma factor 24

heat shock response

membrane protein damage

sigma factor 19

transport of ferric citrate

stages of transcription

1. Initiation

2. Elongation

3. Termination

transcription cycle steps

1. sigma factor directs the RNA polymerase core enzyme to the -35 promoter sequence

2. RNA polymerase denatures a short stretch of DNA at the -10 region, forming an open complex that is stabilized by sigma

3. RNA polymerase core synthesizes RNA, and sigma dissociates from the core after about 12 ribonucleotides have been linked - enters elongation phase

4. Elongation continues until a terminator is encountered - RNA polymerase ceases transcription and the RNA is released

intiation of transcription

1. binding of RNA polymerase holoenzyme to form a closed complex - DNA is still double stranded

2. Sigma binds to the promoter region of DNA

3. RNA polymerase opens DNA helix - forming open complex - transcription begins

4. sigma releases from promoter

5. RNA synthesis continues

consensus sequence

a commonly occurring sequence of nucleotides within a genetic element

elongation of transcription

1. RNA synthesis proceeds in 5' to 3' direction with new ribonucleotides added to the 3' end of the growing chain

2. mRNA is released through exit tunnel

3. Pauses every 100-200 bases - template base slips in the active site and halts enzyme

Why is pausing important for elongation?

allows the enzyme to interact with sequence specific regulatory signals

RNA-DNA hybrid region

region that is formed as RNA is sythesized complementary to the DNA template

U-A rich regions

Rho-independent termination

DNA sequences transcribed into RNA hairpins that stall RNA polymerase

Stretch of Us following a stem loop cause RNA polymerase to pause, stem loop forms, RNA falls off

stem loop

A secondary structure that appears in RNAs consisting of a base-paired region (stem) and a terminal loop of single-stranded RNA.

Both are variable in size

Rho-dependent termination

1. rho protein binds to the rut site in RNA and moves toward the 3' end, following the RNA polymerase

2. RNA polymerase pauses

3. rho protein catches up to the open complex and separates the RNA-DNA hybrid, using its helicase activity