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Flashcards in Module 4 Deck (121):
1

Reverse Transcription

Some viruses are able to make RNA and DNA using RNA. As a template during the process of reverse transcription. (Caveat)

2

Gene

-segment of a chromosome
-contains information for a functional protein or RNA molecule
-element shoes activity can be regulated
-multiple products (proteins and RNA)

3

Genetic information

Information about the primary sequence of gene product

4

Bacterial Genome

One circular chromosome

5

Escherichia length

4639675 Bp - 850x longer than the cell

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Chromosomes

One covalently connected DNA. Molecule and associated proteins
-viral= capsid proteins
-prokaryotic DNA= proteins in nucleoid
-Eukaryotic DNA= proteins in chromatin

7

Karyotype

Constellation of all chromosomes in a somatic cell

8

Telomeres

Tips at the end of eukaryotic linear chromosomes

Needed for successful DNA and cell division

9

Centromere

Sequence of DNA that functions as an attachment point for the mitochondria of mitosis spindle

-key player in cel division
-each chromosome has 1
-region where he two daughter chromosomes are held together during mitosis
-essential for equal distribution of chromosome sets to daughter cell
-contain AT rich repeated sequence

10

Viral genome

Made of DNA/RNA surrounded by protein coat called capsid

11

What fraction of total eukaryotic genome encodes for proteins?

1.5%

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Biological function of non-coding sequence

-DNA regions directly participate in the regulation of gene expression
-some DNA are introns
-Some DNA encodes for small regulatory RNA
- Some DNA junk

13

Exons

Expressed regions of the gene (1.5%)

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Introns

Region of genes that are transcribed but not translated

Removed after transcription

All eukaryotic genes contain introns but bacterial genes do not

15

Ways of DNA compaction

Super coiling in prokaryotes and Histones in eukaryotes

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Non super coiled DNA is called

Relaxed

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Supercooling can be

Negative or positive

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2 forms of supercoiling

Writhing and twisting

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Topoisomers

DNAs that differ only in supercoiling

Same length
Same nucleotide sequence
Different degree of supercoiling

Conversion between topoisomers require DNA strand break
Negatively supercoiled DNA travels faster

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Positive super coiling

Forms in front of RNA polymerase

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Negative super coiling

Forms behind RNA polymerase

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Topoisomerase function

DNA unwinding and rewinding

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Two types of topoisomerase

Type 1 and type II

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Type I

Make transient cute in one DNA strand

Relax DNA By removing negative supercoils

Do not need energy/ATP

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Type II

Make a transient cut in both DNA Strand
Allow passage of one double stranded DNA through the other one

Need energy

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DNA Gyrase

Introduces negative supercoils into baterial circular DNA

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Chromatin

DNA + protein +RNA

material of a eukaryotic chromosome

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Nucleosome

Elementary chromatin units
Forms beads of a string
Consist of DNA wrapped around histones

29

Beads on a string

Beads- 146 bp DNA bound to histone core

String is linker DNA of 54bp bound to histone H1

DNA in a nucleosome forms left handed solenoid

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Structure of nucleosome

10nm in diameter

Histone N terminal tails stuck out sand mediate interactions between nucleosomes

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Nucleosomes assemble into higher order structure

Higher level structures of chromatin depend on chromosomal scaffolds

Appear to involve loops of DNA associated with a protein scaffold

32

Bacterial DNA folding

Supercoiled

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Eukaryotic DNA folding

Wound around positively charged histones to form nucleosomes

34

3 main types of DNA metabolism

DNA replication
DNA repair
DNA recombination

35

3 rules of replication

1. Replication is semi conservative

2. Replication begins at an origin and proceeds bidirectionally

3. Synthesis of a new DNA occurs in the 5’ to 3’ direction and is semidiscontinuous

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Semi-conservative

Rah new DNA has an old parent strand and one new daughter strand

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Replication origin

One or two replication forks proceed bidirectional from the replication origin point

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During DNA synthesis new nucleotides added to the

3’ end (3’OH)

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Leading strand

Made continuously as the replication form advances

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Lagging strand

Made discontinuously in short pieces (Okazaki fragments) and later joined by DNA ligase

41

DNA is degraded by

NUCLEASES

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Exonucleases

Cleave bond that remove nucleotides from the ends of DNA, either 5’ or 3’

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Endonucleases

Cleave bonds within a DNA sequence

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DNA synthesis is catalyze do by

DNA polymerase

45

How many DNA polymerase does EColi have?

4

46

DNA polymerase poker with 2 regions

Insertion site and post insertion site

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Insertion site

Where the incoming nucleotide binds

48

Post insertion

Where the incoming nucleotide binds

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Post insertion site

Where the newly made base pair reside when the polymerase moves forward

50

Processivity

The number of nucleotides added before dissociation of DNA polymerase

51

Deoxynucleoside Triphosphates

Serve as substrates in strand synthesis

52

What is the purpose of the the 2 mg ion sin the active site

3’-OH becomes more powerful nucleophile to attack the alpha phosphate of the incoming nucleotide triphosphate

53

Primer

DNA polymerase needs a base-pair 3’ end of the template- requires a primer

Primer is short DNA/RNA strand complementary to the template

Has a 3’-OH to begin the first DNA polymerase catalyze reaction

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3’ to 5’ exonuclease activity

Responsible for proofreading of mis-incorporated nucleotides and their immediate removals and correction of errors during synthesis

55

DNA polymerase 1

It is abundant but is not ideal for replication

Rate of 600/min is slower than observed replication fork movement

Low processivity
It’s primary function is clean up

In addition to 3’ to5’ exonuclease activity, it has a 5’ to 3’ exonuclease activity. It’s the ability to hydrolyze polynucleotide strands ahead of the enzyme in its path.

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DNA polymerase III

Principle replication polymerase

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DNA Polymerase II, IV, V

Involved in DNA repair

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Replisome

The entire macromolecule complex for DNA replication

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DNA helicases

Uses ATP to unwind and desperate DNA strands

60

DNA topoisomerase

Relieve the stress caused by unwinding

61

DNA binding proteins

Stabilize deprecated strands

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DNA primases

To make RNA primers

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DNA ligases

To seal single stranded breaks

64

Steps of Genomic DNA Replication

1. Initiation
2. Elongation
3. Termination

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Initiation

Starts at origin of replication, where strands of the double-stranded DNA are separated

Regulated to occur only once per cell cycle

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Elongation

Continuous synthesis of the leading strand

Discontinuous synthesis of the lagging strand in strand in short Okazaki fragments

67

What enzyme replaces RNA primer with DNA

DNA Polymerase I

68

What enzyme stitches the gap of the lagging strands?

DNA Ligase

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Mutation

The daughter DNA carries a changed sequence in both strands

Mutations can be substitutions (point mutations), deletions, additions

70

DNA Lesion

DNA Damage

If unrepaired, lesion becomes a mutation

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silent mutation

If mutation has no effect on gene function

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Types of DNA Damage

1. Mismatches
2. Abnormal bases
3. Pyrimidine dimers
4. backbone lesions

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Mismatches

Arise from occasional incorporation of incorrect nucleotides by DNA Polymerase

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Abnormal Bases

Arise from spontaneous deamination, chemical alkylation or exposure to free radicals

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Pyrimidine Dimers

Form when DNA is exposed to UV light

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Backbone Lesions

Occur from exposure to ionizing radiation, free radicals

77

How do repair enzymes “know” which strand is the correct one?

In E. coli, the parent strand is methylated

Dam methylase adds methyl groups to adenines in GATC sequence

Right after the replication and division only the parent strand of DNA is methylated

The newly synthesized strand is unmethylated for a short period of time after the synthesis

78

Base-Excision Repair uses which enzymes

DNA glycosylases

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Base-Excision Repair Mechanism

Detects and removes a specific kind of damaged base.
Uracil glycosylase removes uracil from DNA (C deminates to U and U does not belong in DNA)

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Nucleotide Excision Repair

Repair relies on excinucleases that cleave DNA backbone in two places
Detects and corrects types of damage that distort the DNA double helix.

Fixes Large Distortions in DNA

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Most common examples of biological processes that require DNA recombination?

1.Repair of DNA
2. Segregation of chromosomes during meiosis
3. Enhancement of genetic diversity

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Segments of DNA can rearrange their location

1. Within a chromosome
2. From one chromosome to another

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Types of DNA Recombination

1. Homologous/general recombination
2. Site-specific recombination
3. DNA transposition

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Homologous/general recombination (crossing over)

Exchange between two DNAs that share an extended region of similar sequence

Occurs with high frequency in eukaryotes during meiosis

Assists in DNA repair

Links sister chromosomes to properly segregate them between self and daughter cells

Source of DNA exchange and therefore genetic diversity

85

Site-specific recombination

Exchange only at a particular sequence

86

DNA transposition

Jumping genes – short DNAs that can move from one chromosome to another

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Recombination (crossing over)

occurs in first meiotic division between the chromatids of the homologous chromosomes

Chromatids of the two homologous chromosomes (non-sister chromatids) recombine their segments

88

RNA VS DNA

RNA contains ribose sugar instead of deoxyribose

RNA contains Uracil bases instead of thymine

89

Three main types of RNA molecules

1. Messenger RNAs (mRNAs)
2. Transfer RNAs (tRNAs)
3. Ribosomal RNA (rRNAs)

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Messenger RNAs (mRNAs)

Encode the amino acid sequences of all the polypeptides found in the cell

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Transfer RNAs (tRNAs)

Match specific amino acids to triplet codons in mRNA during protein synthesis

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Ribosomal RNA (rRNAs)

Constitute the ribosome – a molecular machine that is responsible for synthesis of all proteins in a cell

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Transcriptome

Sum of all RNAs

94

Functions of RNA molecules in all living organism

Coding
Replication
3D- Structure Formation
Ligand Recognition
Catalysis
Mechanical function

95

Two main types of RNA Mettabolism

RNA synthesis – transcription

RNA post-synthetic processing

96

RNA synthesis is catalyzed by

RNA polymerases

97

Bacteria contains how many RNA Polymerase

one RNA polymerase

98

Eukaryotes contains how many RNA Polymerase

three RNA polymerases

99

What is a promotor

RNA Polymerase binds to the promoter region to initiate transcription

100

What is a transcription bubble and how long is it

RNA polymerase unwinds DNA-duplex to form transcription “bubble” of ~17 bp

101

In transcription where are new nucleotides added?

New nucleotides are added to the 3’-end of the growing RNA strand

102

DNA Template Strand

serves as template for the RNA polymerase

103

DNA Coding Strand

has the same sequence as the RNA transcript

104

What is the subunit composition of the RNA polymerase holoenzyme?

2 alpha
beta
beta'
W

105

Two alpha sub unit

Function in assembly and bind to UP (upstream promoter) elements

106

Beta subunit

subunit is the main catalytic subunit

107

Beta' subunit

subunit is responsible for DNA binding

108

W subunit

Subunit protects the polymerase from denaturation

109

Bacterial RNA polymerase has an sigma subunit. What is it's function?

Directs enzyme to the promoter

110

Why does RNA polymerase not have proofreading activity?

Lacks 3’ to 5’-exonuclease activity
High error rate of 1/104–1/105

111

Nucleoside triphosphates

serve as substrates in strand synthesis

112

Why majority of genes are regulated at the transcriptional level?

Synthesis of RNA is the first step in gene expression

Synthesis of RNA requires lots of energy

113

Common features of bacterial promoters

Two consensus sequences at positions -10 (TATAAT) and -35 (TTGACA) that are recognized by sigma subunit (also called TATA sequences)

A-T−rich upstream promoter element between -40 and -60 binds the alpha subunit

These sequences govern efficacy of RNA polymerase binding and therefore affect gene expression level

114

Closed Complex (DNA is not unwound)

RNA polymerase binds to a promoter

115

Open complex forms
(Region from -10 to +2 unwinds)

RNA polymerase forms transcription bubble

116

What is sigma subunit replaced by?

Replaced by protein NusA

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Rho Independent Termination

RNA encounters a termination sequence (c and G)
A stable hairpin is formed reducing the length of RNA/DNA
mRNA Released

118

Rho Dependent Termination

Rho helicase binds to the rut site

It migrates along the mRNA and catches up to the RNA polymerase

Separates the mRNA from DNA

119

RNA polymerase I

synthesizes pre-ribosomal RNA

120

RNA polymerase II

is responsible for synthesis of mRNA
-Very fast (500–1000 nucleotides/sec)
-Specifically inhibited by mushroom toxin alpha-amanitin
-Can recognize thousands of promoters

121

RNA polymerase III

transcribes tRNAs, 5S rRNA, RNA components od spliceosome and other small RNA products