Microbial Molecular Biology: Dna Replication

Front 1

Microbial Molecular Biology: Why should you care?

Back 1

If we understand the differences between eukaryotic and prokaryotic molecular biology we can exploit these differences to our benefit (for drugs, vaccines, biotech, etc.)

Front 2

How can we use the differences between eukaryotic and prokarotic molecular biology for our benefit?

Back 2

drugs, vaccines, biotech, etc.

Front 3

What is the central dogma of biology

Back 3

DNA -> RNA -> Protein

Replication -> Transcriptions -> Translation

Front 4

Define Dogma

Back 4

A principle or set of principles laid down by an authority as incontrovertibly true (Oxford American Dictionary)

Front 5

What violated the central dogma of biology?

Back 5

The concept of reverse transcription, where RNA is used as a template to make DNA, violated the dogma. It took a white to catch on.

Front 6

What are the four nucleotide bases of DNA?

Back 6

Adenine (A)

Guanine (G)

Cytosine (C)

Thymine (T)

Front 7

What kind of backbone does DNA have?

Back 7

Deoxyribose sugar + phosphate backbone

Front 8

How are the strands of DNA held together?

Back 8

Strands held together by hydrogen bonds between A+T (2 bonds) and G+C (3 bonds)

Front 9

How many bonds between Adenine and Thymine?

Back 9

2

Front 10

How many bonds between Guanine and Cytosine?

Back 10

3

Front 11

Who was the first person to create an X-ray diffraction image of DNA

Back 11

Rosalind Franklin

Front 12

Who published the double-helix model for DNA and what year?

Back 12

James Watson and Francis Crick published their double-helix model of DNA structure in 1954 (Franklin's data was essential)

Front 13

What is the shape of DNA?

Back 13

DNA strands wind around each other to form a double helix

Front 14

What are the two distinct groves that DNA forms?

Back 14

Forms 2 distinct grooves - a major groove and a minor groove

Front 15

Where do proteins usually interact on a DNA structure?

Back 15

Proteins (usually) interact with the major groove

Front 16

DNA in a living organism typically exists in a "_____/_____" helix

Back 16

right/handed

Front 17

What does a right handed DNA look like?

Back 17

Most DNA molecules, like most screws, are right-handed

Front 18

The DNA double helix is _____

Back 18

Twisted

Front 19

About how many bp per twist in DNA?

Back 19

~10 bp/turn

Front 20

What does introducing or removing additional DNA twists lead to?

Back 20

Supercoiling

Front 21

Define supercoiling

Back 21

Introducing or removing additional twists that imparts strain on the DNA molecule

Front 22

Define positive supercoiling

Back 22

Extra twists (overwinding)

Front 23

Define negative supercoiling

Back 23

Subtraction of twists (underwinding)

Front 24

Most DNA in living organisms is ______ supercoiled.

Back 24

Negatively

Front 25

Supercoiling allows lots of DNA to be packed into a bacterial cell (T/F)

Back 25

True

Front 26

The bacterial chromosome (nucleoid) contains multiple loops held by _____/_____

Back 26

anchoring/proteins

Front 27

What does each loop of chromosome contain?

Back 27

Each loop contains supercoiled DNA

Front 28

How much linear DNA does an E. Coli nucleoid contain?

Back 28

The E. Coli nucleoid contains more than 1mm of linear DNA - at least 400 times longer than the cell itself

Front 29

If E. Coli was the size of Andre the Giant, how long would its DNA be?

Back 29

Its DNA would be more than half a mile long!

Front 30

DNA is wrapped around proteins called _____ to form a nucleosome.

Back 30

histones

Front 31

How much DNA is wrapped around a histone?

Back 31

Wraps around 200 bp of DNA

Front 32

Nucleosomes _____, with higher order folding of DNA, to form _____

Back 32

aggregate

chromatin

Front 33

Supercoiling mediated by DNA gyrase (picture)

Back 33

Front 34

In prokaryotes, _____/_____ ( a class II topoisomerase) introduces _____ supercoils into the DNA

Back 34

DNA/Gyrase

negative

Front 35

What class is DNA Gyrase?

Back 35

A class II topoisomerase

Front 36

What happens when a DNA molecule is exposed to DNA Gyrase?

Back 36

The DNA molecule is broken and resealed on the opposite side of the strand

Front 37

DNA Gyrase is found in both prokaryotes and eukaryotes. (T/F)

Back 37

False. DNA Gyrase found only in prokaryotes

Front 38

What is the net effect of the mechanism of type II topoisomerases?

Back 38

Net effect is insertion of one negative supercoil

Front 39

What makes a good target for antibiotics?

Back 39

1. Inhibition of cell wall synthesis

2. Inhibition of protein synthesis

3. Inhibition of nucleic acid replication and transcription

4. Injury to plasma membrane

5. Inhibition of synthesis of essential metabolites

Front 40

Name 4 antibiotics that inhibit cell wall synthesis

Back 40

penicillin

cephalosporin

bacitracin

vancomycin

Front 41

Name 4 antibiotics that inhibit protein synthesis

Back 41

Chloramphenicol

erythromycin

tetracyclines

streptomycin

Front 42

Name 2 antibiotics that inhibit nucleic acid replication and transcription

Back 42

quinolones

rifampin

Front 43

name 1 antibiotic that hurts the plasma membrane

Back 43

polymyxin B

Front 44

Name 2 antibiotics that inhibit the synthesis of essential metabolites

Back 44

sulfanilamide

trimethoprim

Front 45

name 3 antibiotics that target DNA Gyrase

Back 45

Quinolones

Fluoroquinones

Aminocoumarins

Front 46

Why is DNA Gyrase a good antibiotic target?

Back 46

Not present in most eukaryotic cells = good target

Front 47

Some prokaryotes have reverse gyrase (T/F)

Back 47

True

Front 48

Define reverse gyrase

Back 48

is a topoisomerase that introduces positive supercoils

Front 49

Where is reverse gyrase mostly found?

Back 49

Mostly found in Archaea that grow at extremely high temperatures

Front 50

Why would Archaea that live in high temperatures want reverse gyrase?

Back 50

May protect the DNA from heat denaturation - positive supercoiled DNA is harder to denature

Front 51

What are the characteristics of a prokaryotic chromosome?

Back 51

Circular (usually)

Usually only one

Do not have telomeres

Do not have multiple copies of genes - usually

Greater than 90% of the genome consists of genes encoding for known proteins - very little repetitive sequence

Front 52

What shape is the prokaryotic chromosome (usually)?

Back 52

Circular

Front 53

Do prokaryotics have more than one chromosome?

Back 53

Usually only one

Front 54

Do Prokaryotic chromosomes have telomeres?

Back 54

no

Front 55

How much of a prokaryotic genome consists of genes that encode for known proteins?

Back 55

90%

Front 56

What are the characteristics of a Eukaryotic chromosomes?

Back 56

Linear chromosomes

More than one per cell (23 pairs in humans)

Telomeres on the ends required for complete chromosome replication

Often contain multiple copies of each gene

Contain more DNA than necessary, not all function (or known to be)

<3% of the human genome encodes protein

Front 57

What shape are eukaryotic chromosomes?

Back 57

Linear

Front 58

Why do eukaryotic chromosomes have telomeres?

Back 58

Telomeres on the ends required for complete chromosome replication

Front 59

About how much of the human genome encodes protein?

Back 59

<3%

Front 60

How many bp in Bacterial genomes?

Back 60

0.6-9.4 million bp

Front 61

How many bp in human genome?

Back 61

3 billion bp

Front 62

How much bigger is the human genome vs E. Coli's genome?

Back 62

1000X as large as E. Coli

Front 63

How many more genes does a human genome have vs. E. Coli

Back 63

~6x genes: ~23,000 (human) vs. 4,000 (E. Coli)

Front 64

E. Coli genes use less DNA sequence. (T/F)

Back 64

True

Front 65

How many bp go into a typical bacterial gene?

Back 65

About 1000 bases

Front 66

Define plasmids

Back 66

Plasmids are small circular extrachromosomal units of DNA in bacteria that often encode genes for antibiotic resistance

Front 67

How are plasmids exchanged?

Back 67

Plasmids may be exchanged between bacteria of the same or different species allowing antibiotic resistance to spread rapidly

Front 68

Prokaryotes have Mitochrondria and Chloroplasts. (T/F)

Back 68

False. Eukaryotes have mitochondria and chloroplasts

Front 69

Mitochondria and Chloroplasts contain their own _____ material.

Back 69

Genetic

Front 70

Mitochondria and Chloroplasts replicate _____ of the chromosomes

Back 70

Independently

Front 71

Mitochondria and Chloroplasts have everything necessary for protein synthesis. (T/F)

Back 71

True

Front 72

What do mitochondria/chloroplasts have in order to make them capable for protein synthesis?

Back 72

ribosomes, tRNA, etc.

Front 73

Why are Mitochondria/chloroplasts not independent of the cell?

Back 73

most of their proteins are coded by the chromosomes in the nucleus

Front 74

Mitochondria and chloroplasts are independent of the cell (T/F)

Back 74

False. NOT independent of the cell - most of their proteins are coded by the chromosomes in the nucleus

Front 75

Semiconservative replication (picture)

Back 75

Front 76

DNA replication (picture)

Back 76

Front 77

All ______/______ synthesize in the _____ to _____ direction

Back 77

DNA/Polymerases

5'

3'

Front 78

No known DNA polymerase can initiate DNA synthesis. (T/F)

Back 78

True

Front 79

______ adds an RNA primer to the _____ end

Back 79

Primase

5'

Front 80

In what direction does DNA polymerization occur?

Back 80

5'->3'

Front 81

Only ______ strand is oriented ______ for polymerization to proceed ______.

Back 81

strand

correctly

5'->3'

Front 82

Define leading strand

Back 82

The one strand that is oriented correctly for polymerization to proceed 5' -> 3'

In this strand DNA polymerization is continuous

Front 83

Define the lagging strand

Back 83

DNA polymerization is discontinuous because the strand is not oriented correctly

Front 84

DNA Replication Fork (picture)

Back 84

Front 85

Does does primase do?

Back 85

Primase adds an RNA primer to the lagging strand at multiple points along the strand as it is unwound

Front 86

______/_____ on the lagging strand are later joined together

Back 86

Okazaki/fragments

Front 87

Sealing the fragments on the lagging strand (picture)

Back 87

Front 88

How are the fragments of the lagging strand sealed?

Back 88

DNA polymerase III stops when it reaches a downstream primer

DNA polymerase I has 5' to 3' nuclease activity. It removes the RNA primer and adds DNA

DNA ligase makes the final phosphodiester bond

Front 89

Replication of Circular DNA (picture)

Back 89

Front 90

Origin of replication

Back 90

A specific sequence of approx. 300 bp seen in circular replication

Front 91

Replication is bidirectional (T/F)

Back 91

True

Front 92

What forms during replication of circular DNA

Back 92

Forms theta structures in circular DNA

Front 93

Explain the replisome

Back 93

coordinated synthesis of both strands at once

Contains two DNA polymerase III and other molecules needed for replication

Loops the lagging strand

Replisome is anchored near the midpoint of the cell

Note that the DNA moves, not the replisome!

Front 94

Explain whole-genome sequencing

Back 94

Break genome into thousands of pieces that are easy to sequence

Determine sequence of many short pieces at random

Computer determines sequence overlap to recreate entire genome sequence

Front 95

Whole-genome sequencing (picture)

Back 95

Front 96

How many polymerases do Eukarya have?

Back 96

Eukarya have 3 RNA polymerases

Front 97

How many polymerases do Bacteria and Archaea have?

Back 97

Bacteria and Archaea have one RNA polymerase

Front 98

What are the two forms of Bacterial RNA?

Back 98

core enzyme

Holoenzyme

Front 99

What is the role of the sigma subunit?

Back 99

Role of sigma subunit is to recognize the promoter site on the DNA for initiation of RNA synthesis

Front 100

sigma subunit is tightly bound to the other subunits (T/F)

Back 100

False. Sigma subunit is not tightly bound to the other subunits

Front 101

What does sigma does?

Back 101

Sigma locates the initiation site, then dissociates

Front 102

What do core enzymes do?

Back 102

Core enzyme synthesizes the RNA

Front 103

Define promoter

Back 103

Site recognized by RNA polymerase

Front 104

DNA is unwound and RNA is synthesized from one of the strands. (T/F)

Back 104

True

Front 105

Several different sigma factors - recognize distinct promoters (T/F)

Back 105

True

Front 106

In a single promoter there are ______ sets of ______ that are conserved.

Back 106

2

sequences

Front 107

What are the 2 set of sequences that are conserved in a single promoter?

Back 107

The pribnow box (at -10) - TATAAT

-35 sequence - TTGACA

Front 108

Different sigma factors are used in response to different ______ conditions

Back 108

environmental

Front 109

Name and define E. Coli haas 7 sigma factors

Back 109

70 - "housekeeping" sigma factor, transcribes most genes in growing cells

38 - starvation/stationary phase genes

28 - genes involved in flagellar synthesis

32 - genes involved in heat

24 - genes associated with extracytoplasmic stress

54 - Nitrogen-limitation response genes

19 - genes mediating iron transport

Front 110

Different sigma factors can act as '_____/____'

Back 110

virulence/factors

Front 111

Define unit of transcription

Back 111

The section of DNA that is transcribed into RNA

Front 112

Unite of transcription (picture)

Back 112

Front 113

Can a unit of transcription contain more than one gene?

Back 113

Yes

Front 114

Define polycistronic RNA

Back 114

When there is more than one gene on a unit of transcription

Common in prokaryotes

Front 115

Define operon

Back 115

A group of related genes transcribed as a polycistronic RNA

Front 116

What does Rifamycins do?

Back 116

inhibit bacterial RNA polymerase

Front 117

Define RNA translation

Back 117

Process by which the RNA sequence is converted into protein sequence

Front 118

What does RNA translation use to determine which amino acid comes next?

Back 118

Use the genetic code to determine which amino acid comes next

Front 119

Each set of _____/_____ specifies an amino acid

Back 119

3/nucleotides

Front 120

3 nucleotides are called a ______

Back 120

Codon

Front 121

Define codon

Back 121

set of 3 nucleotides that specify an amino acid

Front 122

How many possible 3 letter codons are there?

Back 122

64 (4^3)

Front 123

Define degeneracy

Back 123

more than one codon codes for an amino acid

Front 124

Define Wobble

Back 124

the 3rd nucleotide can vary

Front 125

Most organisms use what code?

Back 125

The "universal code"

Front 126

Some organisms and organelles use slight variations of the code. (T/F)

Back 126

True

Front 127

Animal mitochondria use the stop codon _____ to encode _____

Back 127

UGA

tryptophan

Front 128

What does animal mitochondria using the stop codon UGA to encode tryptophan an indication of?

Back 128

Indication of different evolutionary history

Front 129

In Mycroplasm (_____) and Paramecium (_____) "_____" (stop) codons can encode _____/_____

Back 129

bacteria

eukarya

nonsense

amino/acids

Front 130

In certain cases ______ codons can encode rare _____/_____ such as _____ and _____

Back 130

nonsense

amino/acids

selenocysteine

pyrrolysine

Front 131

_____ reads the base pairs on the codon

Back 131

tRNA

Front 132

How many nucleotides on transfer RNA?

Back 132

73-93 nucleotides

Front 133

Describe tRNA

Back 133

73-93 nucleotides, chemically modified bases, extensive secondary structure

Front 134

Protein synthesis is mediated by what?

Back 134

Mediated by the ribosome, a ribonucleoprotein complex consiting of ribsomal RNAs and proteins

Front 135

What are the three steps of protein synthesis?

Back 135

1. Initiation

2. Elongation

3. Termination

Front 136

Ribosomes of mitochondria and chloroplasts of eukaryotes are similar to prokaryotic ribosomes (T/F)

Back 136

True

Front 137

How is the initiation of translation started?

Back 137

mRNA, tRNA, plus initiation factors assemble on the 30S subunit

Associates with 50S ribosomal subunit

Front 138

How is the correct reading frame determined for prokaryotes?

Back 138

the 16S rRNA binds to the Shine-Dalgarno sequence, a sequence of 3 to 9 nucleotides just proceding the initiation codon in the mRNA

Front 139

How is the correct reading frame determined in Eukaryotes?

Back 139

Ribosome binds to the 5' cap on the mRNA

Front 140

Define Intiation

Back 140

First charged tRNA binds in P-site (Peptide site)

Front 141

Define elongation

Back 141

A second charged tRNA binds in the A-site (acceptor)

Peptide bond formation occurs between amino acids in P- and A-sites

Front 142

Define translocation

Back 142

Uncharged tRNA in P-site moved to E-site (exit site) and tRNA in A-site moves to P-site

Uncharged tRNA released from E-site and next charged tRNA binds to A-site

Front 143

When does termination of protein synthesis occur?

Back 143

Occurs when the ribosome reaches a stop codon

No t RNA binds

Front 144

______/_____ recognize the stop codon and cleave the attached polypeptide

Back 144

Release/factors

Front 145

During the termination of protein synthesis what does the ribosome dissociate into?

Back 145

Ribosome dissociated into 30S and 50S subunits, which can then form new initiation complexes

Front 146

Define the polyribosome

Back 146

Also known as the polysome

Several ribosomes can translate a single mRNA simultaneously

This speeds up the process and generates additional protein product

Front 147

The polyribosome was first described by whom and what year?

Back 147

The polyribosome was first described by Warner, Knopf & Rich in 1963

Front 148

Why do many antibiotics target the bacterial ribosome?

Back 148

Rely on differences between prokaryotic and eukaryotic ribosomes