Biochemistry Exam 2

Front 1

what are clamp loaders?

Back 1

load the dna clamps

Front 2

What are clamps?

Back 2

help to keep the polymerase in contact w/ the growing strand - Clamps increase processivity, meaning increases the number of nucleotide additions catalyzed by polymerase before it dissociates.

Front 3

What are single stranded DNA binding proteins?

Back 3

bind to ss DNA in order to keep it unwound

Front 4

What are topisomersases?

Back 4

o Topoisomerases mitigate the twisting problem by forming a transient break in the DNA backbone, allowing the DNA to rotate to relieve the additional twists and then resealing break. – RELIEVE TWISTING
o Topoisomerase I- breaks one strand
o Topoisomerase II- breaks both strands

Front 5

What role does RNaseH have in DNA repliction?

Back 5

RNaseH degrades the RNA primer, leaving one ribonucleotide attached to the 5’end of the Okazaki fragment.

Front 6

What role does FEN1 have in DNA replication?

Back 6

FENs catalyse hydrolytic cleavage of the phosphodiester bond at the junction of single and double stranded DNA. FENs can also act as 5'-3' exonucleases on the 5' terminus of the flap strand and on 'nicked' DNA substrates.

Front 7

What are telomeres?

Back 7

the DNS-protein complex that protects ends of chromosomes from attack by nucleoases---6 nucleotide repeats of the sequence TTAGGGShorten each successive cell division and once certain length cell no longer divides.

Front 8

What do telomeres do in DNA replication?

Back 8

play a role in preventing strand shortening during replication.enzyme that catalyzes the addition of telomere is telomerase. mitotic clocks(telomere lenght in most cells inversely related to #of times cells divided

Front 9

What is homologous recombination?

Back 9

Homologous recombination is the exchange of genetic information between identical or nearly identical sequences. The process is initiated when an endonuclease makes a double strand break in a chromatid. An exonucleasethen removes nucleotides to create two protruding 3’ single stranded ends. The 3’ end then finds the homologous region of the second chromatid to begin base pairing. DNA polymerase synthesizes DNA from the free 3’ OH of the invading strand nucleotides in the displaced D loop pair serve as a template for DNA synthesis from the other 3’ end. Nicks are ligated forming two holiday junctions

Front 10

What are holiday junctions?

Back 10

Holiday junctions are points where the strands cross between chromatids. The movement of the crossover point is called the branch migration.

Front 11

What are transposases?

Back 11

Transposeases are enzymes that cleave a transposn form genome and move it to a different location.

Front 12

What are integrases?

Back 12

Integrase is an enzyme that inserts transposable elements into the chromosome.

Front 13

What are 3 classes of transposable elements?

Back 13

DNA only transposon - each end consists of inverted repeats Cut and paste type
b)Retroviral like retrotransposons- direct repeats at each end. Copy and paste type uses reverse transcriptase
c) Nonretroviral retrotransposons- poly A at 3’ end of RNA, 5’ end is truncated. Copy and paste type. Makes reverse transcriptase and moves via an RNA intermediateClass I mobile genetic elements, or retrotransposons, copy themselves by first being transcribed to RNA, then reverse transcribed back to DNA by reverse transcriptase, and then being inserted at another position in the genome. Class II mobile genetic elements move directly from one position to another using a transposase to "cut and paste" them within the genome.

Front 14

What are some types of DNA damage?

Back 14

Deamination -removal of an amine. Cytosine deamination produces uracil, adenine deamination produces hypoxanthine, guanine deamination produces xanthine.

Depurination – cleavage of a purine from the sugar (5,000 events per day/cell).

Alkylation – addition of a methyl group to bases.

Crossing linking of pyrimidines to form dimers. UV light can produce thymine-thymine dimers.

Front 15

What is base excision repair?

Back 15

repairs deaminated, methylated, and oxidized bases. Glycosylase recognizes the damage and removes the base by cleavage of the glycosyl bond. An AP endonuclease then cleaves the 5’ side. DNA polymerase beta has 3’ APlyase activity and cleaves the 3’ side and fills the gap. Ligase seals the nick.

Front 16

What is nucleotide excison repair?

Back 16

repairs things that distort the DNA (TT diamer). Endoonucleases cleave the abnormal chain and remove distorted region. Nicks are made on either side and an oligonucleotide is released. Dna polymerase fills the gap and ligase seals the nick.

Front 17

What is mismatch repair?

Back 17

incorrect base pairing distorts the duplex. Same as nucleotide repair. Recognizes which strand is new (which one to fix) because newly synthesized strands has nicks, parental strand is complete.

Front 18

What is bypass synthesis?

Back 18

Bypass synthesis the polymerase when encountering a lesion just continues. Is done by specific polymerases that are less accurate.
Prokaryotes= DNA polymerase IV and V

Eukaryotes = DNA polymerase η,ί,ξ

Front 19

What are some characteristics of RNA polymerase?

Back 19

­ It catalyzes the addition of nucleotides to a growing RNA chain in the 5’ to 3’ direction.
­ Transcription begins when RNA polymerase binds to the promoter at the start of a gene (initiation).
­ In prokaryotes, a single RNA polymerase synthesizes all of the mRNA, rRNA, and tRNA.
­ RNA polymerases catalyze the addition of nucleotides to a growing RNA chain in the 5’ to 3’ direction.
­ Use DNA as a template.
­ Add nucleotides to the growing RNA strand in a manner similar to DNA polymerases (i.e., transesterification).
­ DO NOT need a primer to initiate RNA strand synthesis.
­ Prokaryotes have a single core RNA polymerase.
­ Eukaryotes have three nuclear RNA polymerases and one mitochondrial RNA polymerase.

Front 20

What is a gene?

Back 20

the basic unit of transcription; A gene is the segment of DNA involved in producing a polypeptide chain. It consists of sequences that precede and follow the coding region, as well as sequences that intervene (introns) between individual coding sequences (exons).

Front 21

What is a promoter?

Back 21

- DNA sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription. In RNA synthesis, promoters mark which genes should be used for messenger RNA synthesis

Front 22

What is initiation?

Back 22

when the RNA polymerase binds to the promoter at the start of a gene

Front 23

What is elongation?

Back 23

sequential addition of nucleotides after initiation

Front 24

What is startpoint?

Back 24

– the first bp transcribed; the promoter is typically in front of the start point

Front 25

What is termination in transcription of prokaryotic genes?

Back 25

transcription is terminated by one of two modes:
i. Rho-independent – stem loop structure of RNA and 6-7 U’s (intrinsic terminator) terminate synthesis. G-C rich stem is proposed to stall polymerase. RNA can fold back on itself forming a GC rich stem plus a loop known as a hairpin.
ii. Rho-dependent – Rho factor participates in termination( has helicase activity which causes release of the RNA

Front 26

Describe a transcription bubble?

Back 26

a molecular structure that occurs during the transcription or replication of DNA when DNA helicase and DNA topoisomerase "unzip" the DNA double strand. DNA polymerase or RNA polymerase may then bind to the exposed DNA and begin synthesizing a new strand of DNA or RNA. As DNA and/or RNA polymerase progresses down the DNA strand in the 3' to 5' direction, more of the DNA double strand is unwound, creating a replication or transcription bubble in the process

Front 27

Eukaryotic RNA polymerases

Back 27

I, localized in the nucleolus and synthesizes rRNA- 28S, 18S, 5.8S

II, located in the nucleoplasm and synthesizes mRNA, snRNA
inhibited by a-amanitin.

III, located in the nucleoplasm and synthesizes tRNA,5S ribosomald inhibited by a-amanitin.

Mitochondrial-contain single rna polymerase that resemble bacteria

Front 28

Prokaryotic RNA polymerases

Back 28

1 rna polymerase synthesizes all of rna except for rna primers(synthesized by enzyme primase) Rifamancin inhibits initiataition of transcription by binding B-subunit of rna polymerase

Front 29

What do promoter consensus sequences do?

Back 29

represent the results of a multiple sequence alignment, where related sequences are compared to each other, and similar functional sequence motifs are found. The consensus sequence shows which residues are most abundant in the alignment at each position.

Front 30

What are types of RNA synthesized by eukaryotic RNA polymerases?

Back 30

rRNA( 28S, 18S, 5.8 S) synthesized from RNA polymerase I

mRNA( also some small nuclear RNAs and snRNA synthesized from RNA polymerase II

tRNA( and 5S ribosomal RNA) synthesized from RNA polymerase III

mitochondrial, all RNA’s

Front 31

What are transcription factors discussed?

Back 31

TFIID starts the assembly process, binding the TATA box via its TBP (TATA-binding protein) subunit.

TFIID binding distorts the double helix, which probably serves as a landmark to denote an active promoter, allowing for subsequence protein assembly steps.

Other TFIIXs assemble upon TFIID.

TFIIH-hyrolyzes ATP

Front 32

Describe processsing of hnRNA

Back 32

primary transcript in eukaryotes that is a precursor to mature mRNA

Process:
- Guanylyl transerase adds 5’ cap
- Poly(A) polymerase- adds A (20-200) to the 3’ end
- Small nuclear- ribonucleoproteins (snRNPs) catalyze splicing
- The 5’ cap is added as RNA is synthesized and serves as a recognition feature in ribosome binding.

Front 33

Describe processing of tRNA

Back 33

Additional nucleotides are cleaved by RNaseP (5’) and an exonuclease (3’)
tRNA nucleotidyltransferase adds the CCA-3’ sequence and other base modifications occur

An endonuclease removes the intron and a mature tRNA is produced following ligation

Front 34

Describe process of rRNA

Back 34

- RNA polymerase I synthesizes three of the four rRNAs in the nucleolus
- These rRNAs are produced as a large 45S precursor
- This precursor is processed by snoRNPs to release 28S, 18S, and 5.8S rRNAs
- 5S is synthesized by RNA polymerase III in the nucleoplasm

Front 35

What is translation?

Back 35

Translation is the process by which the mRNA transcript will be used to determine the amino acid sequence that makes up a protein.

Front 36

What is general structure/fxn of tRNA?

Back 36

transports amino acids to the ribosomes where the amino acids are assembled into polypeptides All have 3’CCA sequences, cloverleaf secondary structure and L-shaped tertiary structure. They have unique regions that recognize and interact with mRNA (the anti-codon) and have specific aminoacyl-tRNA synthtases that couples an amino acid

Front 37

What are codons?

Back 37

A codon is a three- base (nucleotide) sequence that specifies for a particular amino acid . There are 64 possible codons (43 = 64) . 61 of them code for amino acids and 3 code for stops.

Front 38

Know stop and start codons

Back 38

Start codon is AUG. Stop codons include UAG, UGA, UAA

Front 39

What does degeneracy mean?

Back 39

Different triplet combinations code for the same amino acid (some amino acids are coded by multiple codons).
While many amino acids are specified by more that one codon, each codon can only codon for ONE amino acid.

Front 40

What is the wobble hypothesis?

Back 40

mechanism by which tRNA's can recognize more than 1 codon for a specif aa----base at the 5' end of anticodon(the 1st base of anticodon) is not as spatially defined as other 2 bases. Mvmt of that base allows nontraditional base pairing with 3' base of codon(last base of codon) this mvmt is called a wooble...

Front 41

What is the reading frame?

Back 41

An open reading frame starts with an AUG and continues in triplets to a termination codon. 2nd reading frame, the codons are one base off of the AUG register, is blocked with stop codons. 3rd reading frame, the codons are two bases off of the AUG register, is blocked with stop codons.

Front 42

What is a point mutation?

Back 42

a single base change. Transitions- a purine for a purine. Transversions-a purine for a pyridimine.

Front 43

What is silent mutation?

Back 43

a change occurs that specifies the same amino acid. CGA to CGG,

Front 44

What is a misense mutation?

Back 44

– a change that specifies a different amino acid. CGA to CCA,
• Arg to Pro

Front 45

What is a nonsense mutation?

Back 45

– a change that produces a stop codon. CGA to UGA, Arg to stop.

Front 46

What is frameshift mutation?

Back 46

insertion or deletion of a single nucleotide in coding regions. Shifts the reading the frame.

Front 47

What are free ribososmes?

Back 47

Free ribosomes – located in the cytosol and synthesize proteins that remain in the cytosol or are targeted to the nucleus, mitochondria, or other organelles.
o Initiation and elongation begins on free ribosomes
o Proteins synthesized in the rough endoplasmic reticulum have a signal sequence that is recognized by a signal recognition particle (SRP)
o The complex is bound by a docking protein at the ER, then transferred to a translocon (ribosome receptor)

Front 48

What are bound ribosomes?

Back 48

bound to the rough endoplasmic reticulum and synthesize proteins that will be secreted or incorporated into other cellular membranes.

Front 49

What are 3 steps in translation?

Back 49

• Initiation – the reactions that precede the formation of the peptide bond between the first two amino acids.
• Elongation – all the reactions following the formation of the first peptide bond to the addition of the last amino acid.
• Termination – the steps needed to release the completed polypeptide chain and ribosome dissociation.

Front 50

What is prokaryotic initiation?

Back 50

the initiating methionyl T-RNA is formylated, producing a formyl-methionyl tRNA that participates in INITIATION COMPLEX-3 initiation factors---id of initaiting AUG occurs whn sequence in the mRNA-known as Shine Dalgarmo sequence binds to complementary sequence near 31 end of 16S rRNA of small(30S) ribosomal unit

Front 51

What are steps in eukaryotic initiation?

Back 51

1. GTP binds eIF2a
2. Met-tRNAimet binds to form a ternary complex. Note that this initiator tRNA species cannot be replaced with Met-tRNA emet.
3. this complex binds to 40S(small in eukaryote)subunit and occupies what will be the P site.
- eIF-4e binds 5’ cap
-eIF-4a unwind secondary structures
-eIF-4g-binds to eIF-4e
-eIF-4B assists further unwinding
-eIF-3 needed to maintain free 40S subunits, required for 40S subunit with ternary complex to bind to 5’ end
-40S subunit migrates along mRNA to AUG codon
-eIF-5 –GTPases required for 60S joining, release of eIF-2 and eIF-3

Front 52

What are 3 steps in eukaryotic elongation?

Back 52

1. Binding of an aminoacyl-tRNA to the A site (acceptor site) -done by EF-1alpha and GTP is hydrolyzed
2. Formation of a peptide bond between the Met-tRNA i met in the P site (peptidyl site) and the aminoacyl-tRNA in the A site (the formation of the bond is catalyzed by peptidyltransferase).
3. Translocation of the peptidyl-tRNA to the P site (by translocase)

Front 53

Prokaryotic elongation similar except elongation factors are different

Back 53

Prokayotic
First elongation factor EF-Tu
Elongation factor regenerating the first factor EF-Ts
Elongation factor involved in translocation EF-G

Front 54

What is eukaryotic termination?

Back 54

1. A release factor (eRF-GTP) binds when a termination codon (UAG, UAA, or UGA) is encountered and the deacylated tRNA is released from E site.
2. Peptidyl-transferase hydrolyzes the linkage between tRNA and the polypeptide.
3. GTP is hydrolyzed by eRF and the complex dissociates from mRNA as well as the ribosome subunits and deacylated-tRNA

Front 55

What are antibiotics that inhibit translation in bacteria?

Back 55

Tetracycline –interacts with 30S ribosomal subunits and blocks access of aminoacyl-tRNA to A-site of ribosome(mRNA-ribosomal complex)
Streptomycin-binds to 30S subunit and distorts its structure interfering with initiation of protein synthesis. prevents the transition from initiation complex to chain-elongating ribosome and also causes miscoding
Chloramphenicol- blocks the peptidyl transferase reaction on ribosomes
Erythromycin –binds irreversibly to site on 5oS subunit thus inhibiting translocation reaction on ribosomes
Rifamycin – blocks initiation of RNA chains by binding to RNA polymerase (prevents RNA synthesis)

Front 56

What are antibiotics that inhibit translation in both bacteria and eukaryotes?

Back 56

Puromycin – causes the premature release of nascent polypeptide chains by its addition to growing chain end this causing inhibition of further elongation
Actinomycin D- binds to DNA and blocks the movement of RNA polymerase (prevents RNA synthesis)

Front 57

Movement of proteins to correct area of cell after being synthesized

Back 57

Localization signal target cytoplasmic proteins (those make by free ribosomes) to specific cellular locations. These signals are found inspecific areas of the protein and have a specific characteristics (i.e., charge and or sequence)

Proteins made in the ER may be sorted and trafficked to specific cellular domains according to specific signal sequences. The “default pathway” takes a protein through the ER, into the Golgi, and on to the plasma membrane.

Front 58

What is partial proteolysis?

Back 58

amino acid sequences may be removed form a protein during maturation

Front 59

What is phosphorylation?

Back 59

many proteins are modified by a the covalent addition of a phosphate group. Kinases catalyze the addition of a phosphate group to a protein
ATP Phosphates catalyze the removal of a phosphate group from a protein.
Phosphorylation of serine, threonine, or tyrosine residues. May activate, modify, or terminate a protein function

Front 60

What is glycosylation?

Back 60

a process of adding one or more sugars to a protein.
Glycosylation of asparagine, serine, or threonine residues. Alters protein stability, function and solubility
N-linked (asparagine-linked) glycosylation is the most common.

Front 61

What is ubiquination?

Back 61

attachment of small highly conserved protein called ubiquitin to a protein that is defective or destined for rapid turnover are often marked for destruction by ubiquination. They ae rapidly degraded by a proteasome in cytosol.

Front 62

What are other post translational modifications?

Back 62

acetylation, methylation, iodination, palmitoylation, myristolation, ADP-ribosylation…)

Generally:

Fold into unique 3-D conformations
May bind cofactors such as biotin
May undergo posttranslational modifications
May be sorted to specific cellular domains or organelles
Attachment of lipids can help anchor proteins in membranes.

Front 63

What is a bacteria operon?

Back 63

The structural genes that code for proteins involved in particular metabolic pathway are often fuound sequentially grouped on chromosome along with cis acting regulatorty that determine trnscription of these genes.a unit of bacterial gene expression and regulation that consists of the following:Structural Genes, Regulatory Genes and control elements.

Front 64

What are structural genes?

Back 64

encode for anything other than a regulator protein, that are coordinately regulated. Meaning, they are transcribed to form a polycistronic mRNA.

Front 65

what are regulatory genes?

Back 65

code for regulator proteins. They encode for mRNA as well, which will make regulator proteins.

Front 66

What are control elements?

Back 66

sit on top of structural gene cluster and control whether or not those genes are transcribed. A particular DNA sequence in control element is called the operator, where a particular regulatory protein, the repressor, binds

Front 67

Describe regulation of lac operon

Back 67

the lac-operon codes for 3 proteins involved in catabolism of dissacharide
Lactose operon consists of 3 structural genes (lacZ, lacY, & lacA), a repressor gene (lacI), and control elements (lacP = promoter, lacO = operator, CAP (catabolite activator protein), and CAP binding site)
i. The repressor gene, lacI, is always transcribed and has a high affinity for the operator, this causes RNA Poly to not always be able to bind. (It is able to bind rarely when the repressor leaves, and the binding site to the operator is open for a split second)
ii. When an inducer (allolactose) is present, it binds to the repressor and reduces its affinity causing transcription to occur.
iii. Mutations can inhibit induction by preventing inducer binding to the repressor or permanently turn on the structural genes (lacZ, lacY, & lacA) by preventing repressor binding.

Lactose operon is generally turned off unless it is induced

Front 68

Describe operation of tryptophan operon

Back 68

Tryptophan operon is generally turned on unless it is repressed

Tryptophan operon has 5 structural genes to code for 3 enzymes, its synthesis can be regulated at the transcriptional level and enzyme level, through feedback inhibition affecting anthranilate synthetase.
i. trpR gene encodes for the tryptophan repressor which binds with tryptophan, a corepressor that increases its affinity greatly to the operator.
ii. When the corepressor is not bound, RNA Poly is able to bind to the promote
iii. Using attenuation, when tryptophan levels are low, tRNAtrp will be low and the ribosomes stall in the leader mRNA sequence. This induces a secondary structure to signals for RNA Poly to continue past the attenuator.

Front 69

What is an inducer?

Back 69

stimulate induction, which is when the transcription of genes in an operon increases in response to a substrate.

Front 70

What is a corepressor?

Back 70

stimulate respression, which is when the transcription of genes in an operon decreases in response to a substrate.

Front 71

What is catobolite repression?

Back 71

When glucose is present it causes the transcription of the lactose operon to not be induced even in the presence of lactose. This is due to AC (adenyl cyclase) being inhibited by glucose and not allowing cAMP to be made, which in turn cannot bind CAP. And CAP binds the catabolite activator protein.

Front 72

What are attenuators?

Back 72

intrinsic terminators located at the beginning of a transcriptional unit, which reads the RNA polymerase to decide if it should continue transcription or terminate.

Front 73

What is feedback inhibition and how does it regulate enzymatic activity?

Back 73

when the level of an enzyme or product causes further products not to be synthesized
Tryptophan levels increases bind via feedback inhibition and can shut level down

Front 74

What levels may eukaryotic gene expression be regulated?

Back 74

transcriptional control
i.For most genes, transcriptional control is the leader for regulating expression.
RNA processing control-
RNA transport and localization control
translation control-
mRNA degredation contol- protein activity control-

Front 75

what is histone acetylation?

Back 75

o Acetylation of lysines will weaken the interactions between the + charged histones and the – phosphate groups of the DNA backbone. This may lead to gene transcription b/c transcription factors have access to DNA and then recruit RNA Poly II.

Front 76

What is DNA methylation?

Back 76

C in CG can be methylated, and if it occurs before replication, then it is still methylated afterwards. CG-regions are found in gene promoter regions. Transcription factors can freely associate with gene promotor regions if the C’s are NOT methylated. Methylation causes gene inactivation.
Methylation of DNA and deacetylation of histones often occur together to repress transcription of a particular gene

Front 77

Describe imprinting

Back 77

o Imprinting- a difference in behavior between alleles inherited from each parent.
This can be caused by a specific pattern of methyl groups being inherited by germ cells.

Front 78

Angelman syndrome

Back 78

Severe mental retardation, with absence of speech
Seizures
Jerky gait
Inappropriate laughter
Protruding tongue
Enlarged jaw

Front 79

Prader-Willi syndrome

Back 79

Mild to moderate mental retardation
Hypotonia (abnormally low muscle tone) and poor feeding in infancy
Short stature
Small hands and feet
Small external genetalia
Hyperphagia (compulsive overeating) and massive obesity beginning at about two years old

Front 80

components of the basal transcriptional apparatus

Back 80

TFIID binds DNA when TATA box is accessible. Once TFIID binds, other transcriptional factors bind in an ordered manner (e.g. TFIIB, TFIIA) to form the preinitiation complex. Then RNA Poly II is phosphorylated by TFIIH and transcription now begins. These transcription factors together with RNA Poly II make up the basal transcriptional apparatus, which is needed to transcribe any promoter for RNA Poly II.

Front 81

What are general (basal) factors?

Back 81

transcription factors that bind the core promoter elements to recruit and position RNA poly II

Front 82

What are activators?

Back 82

transcription factors that bind to proximal or enhancer elements to facilitate general factor assembly, which will influence the frequency of initiation, and thus increase gene expression. Main function is to attract, position, and modify the general transcription factors and RNA Poly II so that transcription can begin.

Front 83

What are Inducible Factors ?

Back 83

a transcription factor that’s activity is regulated (e.g. ligand binding)

Front 84

What are Repressors?

Back 84

transcription factors that bind to consensus sequences in the promoter to prevent general factor assembly / inhibit gene expression. They inhibit by recruiting repressive chromatin remodeling complexes and recruiting histone deacetylases.

Front 85

What are coactivators?

Back 85

Some activators recruit enzymes that alter chromatin structure - these enzymes are referred to as co-activators, proteins that do not bind DNA themselves, but assemble on other DNA-bound gene regulatory proteins.

Front 86

What is Histone acetylase ?

Back 86

enzyme involved in acetylation of histones( important in activation of chromatin for transcription)

Front 87

What is Chromatin remodeling complex?

Back 87

alters the positioning of nucleosomes and allows transcription factors access to specific regions of dna

Front 88

What is a mediator?

Back 88

an important co-activator that is required for both basal and regulated transcription at promoters used by RNA pol II and stimulates the phosphorylation of the CTD by TFIIH
• Mediator is a protein complex consisting 20 or more subunits that binds tightly to the carboxyl-terminal domain of the largest subunoit of Pol II

Front 89

What does a mediator do?

Back 89

• It binds RNA pol II and some activators
It stimulates phosphorylation ofRNA pol II by TFIIH, allowing transcription to begin.

Front 90

What are Proximal elements?

Back 90

consensus sequences that are near the transcriptional start point and are upstream.

Front 91

What are Enhancer elements? either orientation

Back 91

cis-acting DNA sequences that increase rate of initiation of transcription by RNA polymerase II. They can be located upstream (to the 5' side) or downstream (3' side) of transcription site
2) they can be close to or thousnads of bps away from the promoter and can occur on either strand of dna

Front 92

What are steroid hormone receptors?

Back 92

Specific response elements are bound by specific receptors (e.g., the glucocorticoid receptor binds to the glucocorticoid response element).
Streroid hormone receptors contain the following:
A ligand-binding domain; where the steroid binds the receptor.
A DNA-binding domain; where the receptor binds the response element.
A transactivation domain; the domain that binds co-activators.
A nuclear localization signal (NLS); directs the receptor to the nucleus at the appropriate time.
These receptor-ligand complexes accumalate in nucleus, dimerize, and bind to specific regulatory dna sequences(HRE-hormone response elements) in association with co-activators thereby causing promoter activation and increased transcription.

Front 93

What role do they play in eukaryotic gene regulation?

Back 93

Steroid hormones, and other lipophilic hormones, activate or inhibit gene transcription by binding nuclear receptors.
These receptors bind to hormone-response elements (short consensus sequences of DNA) at genes that possess them. These receptors, and their response elements, allow for rapid changes in gene expression in response to changes in hormone levels.

Front 94

What is mechanism of glucocorticoid?

Back 94

Glucocorticoid receptors (GR) are retained in the cytoplasm bound to heat-shock proteins (HSP) (A).
In the presence of cortisol, which binds to GR, the nuclear localization signal sequence is exposed and the GR dimerizes (forms a homodimer) (B).
The GR dimer binds to the glucocorticoid response element (GRE) of genes possessing it (C).
The dimer recruits activators to influence the transcription of the genes possessing the GRE (D).

Front 95

What is alternative splicing?

Back 95

A particular mRNA transcript may be spliced in more than one way, thereby allowing the same gene to produce a corresponding set of different proteins.
– This phenomenon is referred to as alternative splicing and ~75% of genes in humans are alternatively spliced.
• Alternative splicing can be regulated.
– It can be negatively regulated by preventing the splicing machinery from gaining access to a particular splice site on the RNA. In this case, a repressor protein binds and blocks the access of the splicing machinery.
– It can be positively regulated by helping direct the splicing machinery to an otherwise overlooked splice site. In this case, an activator protein binds to attract the splicing machinery to a particular splice site.

• Alternative splicing can also be constitutive.
– Constitutive alternative splicing occurs because there is an intron sequence ambiguity.
• The spliceosome is unable to clearly distinguish between two or more alternative pairing of 5’ and 3’ splice sites.
• Consequently, different choices are made by chance on different transcripts.
• This results in several versions of the protein encoded by the gene being made in all cells expressing it.

Front 96

What is ALTERNATIVE CLEAVAGE?

Back 96

• Remember that the 3’ end of a eukaryotic mRNA is formed by cleavage.
• A cell can control the site of this cleavage and therefore can change the C-terminus of the resultant protein.
• An example of this is the switch from the synthesis of membrane-bound to secreted antibody of B lymphocytes. At first, a B lymphocyte produces an antibody that is anchored to the plasma membrane where it serves as a receptor for antigen. Antigen binding causes the cell to secrete its antibody. The secreted antibody is identical to the membrane-bound form except at the extreme C-terminus.

Front 97

What is RNA STABILITY ?

Back 97

mRNA stability influences the level of gene expression. How long and mRNA remains in cytosol influences how much protein product can be produced form that messenger.
• The IRE-BP also recognizes IREs in the 3’-UTR of the mRNA encoding the transferrin receptor.
• In the absence of iron, IRE-BP is bound to the IRE.
• Binding of the IRE-BP blocks access to a endonuclease site, and the mRNA is quite stable and is translated.
• Increased concentrations of iron promote the dissociation of IRE-BP from the 3’-UTR, exposing the endonuclease site.
• The mRNA is then rapidly degraded and translation of the transferrin receptor ceases

Front 98

what is RNA EDITING?

Back 98

RNA editing alters the nucleotide sequence of an RNA transcript once it is synthesized and changes the message it carries.
• Apolipoprotein B undergoes a C-to-U edit in the small intestine, creating a premature stop codon that produces a short form of the protein.
• In the liver, full-length apolipoprotein B is produced.
• The two protein isoforms have different properties, and each plays a specialized role in lipid metabolism that is specific to the organ that produces it.

Front 99

What are Proto-oncogenes: (Cyclin, Cdk)?

Back 99

A class of genes for which a gain-of-function (GF) mutation pushes a cell towards cancer---Proto-oncogenes code for proteins that help to regulate cell growth and differentiation. Proto-oncogenes are often involved in signal transduction and execution of mitogenic signals, usually through their protein products. Upon activation, a proto-oncogene (or its product) becomes a tumor-inducing agent, an oncogene.[7] Examples of proto-oncogenes include RAS, WNT, MYC, ERK, and TRK.

Front 100

What are Oncogenes?

Back 100

Mutant over active form of proto-oncogenes; act in dominant manner

Front 101

What are Tumor suppressor genes?

Back 101

Class of genes for which a loss-of-function (LF) mutation drives a cell towards cancer; act in a recessive manner

Front 102

What is Overactive mutation (GF) ?

Back 102

Mutation in a SINGLE copy of the Proto-oncogene creates oncogene. Activation (Act) leads to Proliferation (Prolif)

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What is Underactive mutation (LF)?

Back 103

Mutation occurs in BOTH copies of gene. Act & Prolif.

Front 104

What is a Tumor AKA neoplasm?

Back 104

Growing mass of abnormal cells that results from unrestrained cell %
o Benign: mass remains clustered
o Malignant: cells acquired ability to invade surrounding tissue

Front 105

What is Cancer?

Back 105

Malignant tumor; mutations in somatic cells, proliferates and invades

Front 106

What is Metastasis?

Back 106

Cells ability to break loose, enter blood stream & lymph tissues; form secondary structures---can break away, leak, or spill from a primary tumor, enter lymphatic and blood vessels, circulate through the bloodstream, and be deposited within normal tissue elsewhere in the body.

Front 107

classes of oncogenes What is Growth Factor?

Back 107

regulate growth by serving as ligands that bind to cellular recptors on plama membrane----this stimulates a signal transduction pathway that activates transcription of certain genes

Front 108

classes of oncogenes -What are Growth Factor Receptors?

Back 108

receptor found on cell surfaces that growth factors bind to
*GF & GFR can lead to increased signal transduction and abnormal cell proliferation

Front 109

classes of oncogenes- What are Signal Transducers?

Back 109

o intracellular components
• Example: GTP turns Ras on; hydrolysis turns Ras off- mutation to the
• hydrolysis can lead to abnormal cell proliferation

Front 110

classes of oncogenes -What are Mutations?

Back 110

increase transcription factor production/activity leads to inappropriate cell division

Front 111

classes of oncogenes-What are DNA-binding proteins?

Back 111

in nucleus (Transcript-factors)

Front 112

classes of oncogenes-What are Regulatory Molecules?

Back 112

(Cyclins, Cyclin-Dep Kinases, Kinases Inhibitors)
• oncogenes inappropriate activity generally leads to increased cell proliferation!!!

Front 113

describe the control system for the cell cycle

Back 113

CC process includes DNA, replication, mitosis, and cytokinesis
Cyclin-dept kinases (Cdks): kinases rise & fall during cell cycle; when active, they phosphorylate
intercellular proteins that initiate or regulate major events of cell cycle and produce orderly division.
Cyclins: protein that regulates Cdk activity; undergo cycles of syn & degradation

Front 114

Describe the way Cdk and cyclin oncogenes lead to increased cell proliferation.

Back 114

Cdk: associated with different cyclins to trigger different cell cycle events
oActivity of Cdk terminated by cyclin degradation (breakdown)
S-cyclin binds to Cdk= S-Cdk complex; triggers S-phase of Cell Cycle
S-Cdk triggers DNA replication machinery
Cyclin: rise & fall is primary determinant of Cdk activity; regulatory mechanisms fine tune Cdk activity
Phosphor (by kinase) & dephosphorylates (phosphatase) can control complex activity
Cyclin isn’t enough to activate complex, additional cyclins needed

-G1 (Cyclin D) w/ Cdk-promotes start of cell cycle
G1/S-cyclin (Cyclin E) w/Cdk-commit cell to DNA replication
-S-cyclin (Cyclin A) w/Cdk- required for initiation of DNA replication
M-Cyclin (Cyclin B) w/Cdk- promotes mitosis

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What is apoptosis?

Back 115

programed cell death; cell shrinks, chromatin condenses, nucleus fragments, & cell breaks up

Can be initiated by DNA damage, lack of necessary GF, or presence of death signaling proteins w/in environment
Resistance to apoptosis is key to malignant cells b/c enables them to increase in # and survive

Front 116

What are mechanisms of apoptosis?

Back 116

Intrinsic: mitochondrial pathway: mito proteins (cytochrome c) from intermembrane space enter cytoplasm
Extrinsic: death receptor pathway; response to binging of a death ligand (TNF) to death receptor
o Both pathways converge on caspases, enzymes that cleave cellular proteins; which are activated by proteolytic cleavage-(2 grps: 1-initiator~cleave other procaspases & act; 2-execution~other cellular proteins invol. maintaining cellular integrity)

Front 117

what role the Bcl-2 gene family plays in apoptosis?

Back 117

Bcl-2 family members integrate pro-death & anti-death signals to determine whether the cell should commit suicide
Anti-apoptotic-prevents a death signal from initiating apoptosis
Bid & Caz are Ccl-2 family members that are pro-apoptotic
When mutated & oncogenic it is over-expressed; disrupts the regulation of pro- & anti- factors

Front 118

What is the normal role of Rb tumor suppressor?

Back 118

inhibit cell proliferation in response to certain signals (DNA damage); if not there ultimately leads to cancer
Rb (Retinoblastoma): rare intraocular malignant neoplasm of infants; cytochrome 13
When not phosphorylated it binds E2Fs, blocking G1/S transition
When phosphorylated it releases E2Fs and the blockade is released-transition occurs
o

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What is normal role of p53 tumor suppressor?

Back 119

They inhibit cell proliferation in response to certain signals (DNA damage); if not there ultimately leads to cancer
P53: Phosphorylation by kinase leads to an increase in its number & activity;
o Stimulates the tf p21 which binds to the G1-Cdk complex and prevents the phosphorylation of Rb-inhibits entry into the S phase.
o Stimulates GADD45-DNA damage enzyme-combined activity; this inhibits the transition to the S phase & transcribing a DNA repair enzyme (gives cell opportunity to fix damage-if repaired decrease of p53 activity & if not-increase signaling for apoptosis to occur

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What is NF-1?

Back 120

• NF1 is the most common neurofibromatosis, occurring in 1 in 3,000 to 4,000 individuals in the United States.
–NF-1 is a tumor suppressor gene that regulates the activity of Ras.
– It does so by behaving as a GTPase-activating protein.

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What disorders related to NF1?

Back 121

This disorder affects the nervous system and causes tumors to grow around nerves.
– Tumors begin in the cells that make up the myelin sheath and often spread into adjacent areas (neurofibromas).
– Other symptoms include
• Malformation of the long bones and curvature of the spine (scoliosis)
• Tumors on the optic nerve which can cause visual loss
• High blood pressure and other blood problems
• Tumors on the spine and brain: increase risk of epilepsy
• Tumors or lesions on the gastrointestinal
• Hearing defects

• Individuals with NF1 have a mutation in the gene that encodes NF-1.

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What is APC?

Back 122

regulatory protein and tumor supressor gene. When it is bound to b-catenenin it inhibits cell proliferation.Mutatations in APC are found in vast majority of sporadic colon cancer.

Front 123

What disorders related to APC?

Back 123

Familial adenomatous polyposis coli (FAP) is a hereditable colorectal cancer, in which hundreds or thousands of polyps appear early in adult life (see B).
– If they are not removed, one or more will almost always progress to become malignant.
• 12 years is the average time from the first detection of polyps to the diagnosis of cancer.
• Individuals with FAP have mutation in, or deletion of, the tumor suppressor gene adenomatous polyposis coli (APC).

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What is deoxyribose?

Back 124

pentose sugar
no hydroxyl group (OH) at C2
when added to a base, C atoms are numbered 1’ to 5’

Front 125

What is structure of 3', 5' phosphodiester bond b/t nucleotides of polynucletide chain?

Back 125

Nucleotides are covalently linked together by 3’,5’-phosphodiester bonds.
b. 5’ end has free 5’ carbon –
c. 3’ end has 3’ nucleotide carbon – free
d. 3’ to 5’ phosphodiester bond – do not hydrolyze frequently – bad if want to form process

Front 126

What is base pairing b/t complementary strands of DNA?

Back 126

Adenine to Thymine with 2 hydrogen bonds
Cytosine to Guanine with 3 hydrogen bonds

Front 127

Describe structure of DNA

Back 127

a. Two polynucleotide chains coiled around a common axis.
b. Chains are paired in an antiparallel manner.
c. In B-DNA bases are stacked inside (hydrophobic), perpendicular to the axis. Deoxyribose-phosphate backbones (hydrophilic) are on the outside.
d. Phosphate groups give DNA a net negative charge.
e. Bases are major groove
f. Minor groove– deoxyribose and 3-5 phosphodiester linkages

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What are 3 forms of DNA?

Back 128

A, B, and Z

Front 129

What is deoxyribose?

Back 129

pentose sugar
no hydroxyl group (OH) at C2
when added to a base, C atoms are numbered 1’ to 5’

Front 130

What is ribose?

Back 130

pentose sugar
hydroxyl group in C2 position

Front 131

What are purines?

Back 131

adenine and guanine – 9 member ring
bound at N9 and C1’ of deoxyribose

Front 132

What are pyridimines?

Back 132

cytosine, thymine, uracil (RNA) – 6 member ring
bound at N1 and C1’ of deoxyribose

Front 133

What is a nucleoside?

Back 133

glycosidic linkage between a base and deoxyribose
ie deoxy to adenine=deoxyadenine

Front 134

What is a nucleotide?

Back 134

nucleoside is linked to a phosphate group –
Nucleotides consist of a phosphate ester, a pentose sugar, and a heterocyclic base which are covalently bonded together

Front 135

What is structure of 3', 5' phosphodiester bond b/t nucleotides of polynucletide chain?

Back 135

Nucleotides are covalently linked together by 3’,5’-phosphodiester bonds.
b. 5’ end has free 5’ carbon –
c. 3’ end has 3’ nucleotide carbon – free
d. 3’ to 5’ phosphodiester bond – do not hydrolyze frequently – bad if want to form process

Front 136

What is base pairing b/t complementary strands of DNA?

Back 136

Adenine to Thymine with 2 hydrogen bonds
Cytosine to Guanine with 3 hydrogen bonds

Front 137

Describe structure of DNA

Back 137

a. Two polynucleotide chains coiled around a common axis.
b. Chains are paired in an antiparallel manner.
c. In B-DNA bases are stacked inside (hydrophobic), perpendicular to the axis. Deoxyribose-phosphate backbones (hydrophilic) are on the outside.
d. Phosphate groups give DNA a net negative charge.
e. Bases are major groove
f. Minor groove– deoxyribose and 3-5 phosphodiester linkages

Front 138

What are 3 forms of DNA?

Back 138

A, B, and Z

Front 139

What are characteristics of A-DNA?

Back 139

A-DNA – 11 bp per turn – low humidity – high salt right handed

Front 140

What are characteristics of B-DNA?

Back 140

B-DNA – 10 bp per turn – high humidity – low salt right handed
i. average overall structure in living organisms

Front 141

What are characteristics of Z-DNA?

Back 141

Z-DNA – 12 bp per turn – left handed

Front 142

What are components of nucleosome?

Back 142

a. Nucleosomes are regularly repeated units – made of DNA wrapped around histones
b. They are composed of an octameric histone cluster containing two each of H2A, H2B, H3, and H4.
c. DNA (147 bp) is wrapped around the octamer and makes contact with histones at the minor groove.
d. ~200 bp of DNA is associated with each nucleosome (DNA wrapped around core + linker DNA).

Front 143

What is hiearchy of DNA packaging?

Back 143

1.Nucleosome
2.10nM fiber – beads on a string – many nucleosomes joined (3-6x shorter)
3.30nM fiber of solenoid (6-7 nucleosomes per turn – 40x shorter)
4.Loops – (1000x shorter) – formed by topoisomerases – avg packaging during interphase
5.Chromosome – 10000x shorter
Linker and 147 pairs – 8 octomer – 10nM fiber – 30nM fiber (6-7nucleosome, 40x) – scaffold (solenoid 1000x) – condensins (10000times)

Front 144

What is heterochromatin?

Back 144

highly condensed chromatin, that is not transcriptionally active.

Front 145

What is euchromatin?

Back 145

less condensed chromatin, that is transcriptionally active.

Front 146

Describe basic structure of eukaryortic chromosome?

Back 146

Each haploid cell (i.e., egg or sperm) contains 1 copy of chromosomes 1-22 (autosomes) and either an X or Y.
Diploid cells contain 2 copies of chromosomes 1-22 and either 2 X (females) or an X and Y (male).
A karyotype is an organized profile of chromosomes.
The human genome contains ~6 billion base pairs and encodes ~25,000 genes.
G band – lower GC content
R band – high GC

Front 147

3 sequences needed to replicate chromosomes

Back 147

Telomere – ends would be shortened and info lost
Origin of replication – sequence necessary to replicate DNA
Centromere – area where mitotic spindle assembles – where the sisters line up

Front 148

What is replication?

Back 148

DNA serves as a template for its replication and, consequently, plays an essential role in heredity.

Front 149

What is transcription?

Back 149

Genetic information contained in DNA is converted to RNA.

Front 150

What is translation?

Back 150

RNA is converted to protein

Front 151

What is tRNA?

Back 151

Transfer RNA (tRNA): activates amino acids and recognizes codons in mRNA. It carries its specific aa-covalently attached to its 3' end-to site of protein synthesis.

Front 152

What is rRNA?

Back 152

Ribosomal RNA (rRNA): structural and catalytic components of the ribosome.
central component of the ribosome, the protein manufacturing machinery of all living cells. The function of the rRNA is to provide a mechanism for decoding mRNA into amino acids and to interact with the tRNAs during translation by providing peptidyl transferase activity

Front 153

What is mRNA?

Back 153

Messenger RNA - carries genetic information from the genome to ribosomes where it is used as template for protein synthesis.

Front 154

What is role of mRNA 5'cap?

Back 154

Principle role is to serve as a recognition feature for initiation factor binding.
Eukaryotic mRNAs lacking a cap are not translated efficiently in in vitro systems.

Front 155

What is role of 3"tail?

Back 155

i. The poly(A) sequence IS NOT coded in the DNA, but is added to mRNA in the nucleus after transcription.
ii. Its addition is catalyzed by the enzyme poly(A) polymerase (20-200 A’s).
The poly(A) tail is useful in isolating mRNA
Plays a role in stabilizing mRNA.
Contributes to the initiation of translation.

Front 156

Prokaryotic and eurkaryotic mRNA differences

Back 156

mRNA carries information (as a sequence of codons) for the synthesis of one or more proteins.
Monocistronic-information for one protein (eukaryotes).
Polycistronic- information for more than one protein (prokaryotes).
Eukaryotic include long sequences of adenine nucleotides(a "poly-A tail")
on 3'-end of RNA chain, plus "cap" on 5'end consisting of 7-mehthylguanosine attached backward

Front 157

Describe Prokaryotic ribosmes

Back 157

Prokaryotes are 70s – made of a 30s and 50s subunit-50S (5S RNA and 23S RNA) and 30S (16SRNA)

Front 158

Describe Eurkaryotic ribosomes

Back 158

Eukaryotes are 80s – made of a 40s and 60s subunit--60S ( 5S, 28S, 5.8), 40S( 18S)

Front 159

Describe structure of tRNA

Back 159

Amino acids are bound to tRNAs.
tRNA recognizes specific mRNA sequences (codons) to incorporate the correct amino acid residue. It serves as the intermediary between genetic information and protein synthesis.
Two important features, CCA-3’ sequence and the anticodon triplet.
tRNAs are 74-95 nucleotides long and have both secondary and tertiary structure.
Cloverleaf – secondary structure
Can also do tertiary structure

Front 160

Describe mechanism of zidovudine

Back 160

ZDV inhibits the production of DNA from viral RNA by preventing the elongation of a DNA strand by reverse transcriptase once it has been incorporated. Notice that ZDV does not have the necessary 3’-OH group for the synthesis of a 3’,5’ phosphodiester linkage. Therefore, the viral DNA is not made and it can not be integrated into a host chromosome.
Nucleotide has high affinity for reverse transcriptase
Does not inhibit dna polymerase

Front 161

Describe mechanism of 5-fluorouracil

Back 161

The chemotherapy agent 5-FU (fluorouracil), which has been in use against cancer for about 40 years, acts in several ways, but principally as a thymidylate synthase inhibitor. Interrupting the action of this enzyme blocks synthesis of the pyrimidine thymidine, which is a nucleotide required for DNA replication. Thymidylate synthase methylates deoxyuridine monophosphate (dUMP) into thymidine monophosphate (dTMP).

Front 162

What does semiconservative mean?

Back 162

• DNA replication is semiconservative.
• The polynucleotide strands are separated from each other and used as templates for the synthesis of complementary daughter strands.
• Each new strand has one original template

Front 163

What does template do?

Back 163

provides the sequence information

Front 164

What does a primer do?

Back 164

provide a free 3’-OH group for the addition of nucleotides to the strand being synthesized

Front 165

What do precursors do?

Back 165

Deoxynucleoside 5’-triphosphates??

Front 166

What do DNA polymerases do?

Back 166

catalyzes the formation of the 3’,5’ phosphodiester bond beween nucleotides....an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best-known for their role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand.

Front 167

What are sliding clamps?

Back 167

keep DNA polymerase in contact with the growing chain

Front 168

What do helicases do?

Back 168

separate DNA strands by unwinding at replication fork-energy for this fxn provided by ATP

Front 169

What are primases?

Back 169

enzymes which synthesize primers (normally short segments of RNA)

Front 170

What do ss DNA binding proteins do?

Back 170

keep template strands separated

Front 171

What are nucleases?

Back 171

catalyse 3’5’ phosphodiester bond thus hydrolyzing nucleic acids – includes endonucleases and exonucleases

Front 172

What are ligases?

Back 172

seals nicks created during DNA synthesis using AMP

Front 173

What is fxn of DNA polymerase?

Back 173

The function of DNA polymerase is to synthesize complementary bases into the daughter strand from the template strand in a 5’ to 3’ direction. DNA polymerase also proofreads the daughter strand to ensure accuracy

Front 174

What are some prokaryotic dna polymerases?

Back 174

Pol III – used for the synthesis of leading strand and the synthesis of most Okazaki frags
Pol I - used to fill the gaps once the primer is removed

Front 175

What are some eukaryotic dna polymerases?

Back 175

Pol a/primase b- used for priming
Pol δ- synthesis of the leading strand
Pol η- damage bypass less specific than Pol δ

Front 176

What is the overall accuracy of a dna polymerase?

Back 176

Using fit, proofreading, and strand-directed mismatch repair, only 1 error is made for every 1 billion nucleotides incorporated!
Therefore, only ~6 errors are made for every round of DNA replication in a human diploid cell

Front 177

What is the replication fork?

Back 177

the point where the double stranded parental DNA separates allowing replication to proceed

Front 178

What is an Okazaki fragment?

Back 178

the small sections of DNA that compose the daughter side of the lagging strand

Front 179

What is the leading strand?

Back 179

is the daughter strand of DNA that is continuously synthesized from 5’ to 3’

Front 180

What is the lagging strand?

Back 180

is composed of discontinuous bands of double stranded DNA due to DNA polymerase synthesizing in a 5’ to 3’ direction

Front 181

Where is DNA replication initiated in Prokaryotes?

Back 181

the ori C is the site where dna replication is intitiated. It is bound by Dna A which causes separation of the strands. Dna C then binds which allows Dna B to bind which is a helicase

Front 182

Where is DNA replication initated in eukaryotes?

Back 182

Eukaryotes have multiple sites along the DNA helix(origins of replication) This provides a mechanism for rapidly replicating the great length of dna molecules. Many sites where replication begins spaced 50,000 -100,000 bp apart. Ori extends bidirectionally until it comes in contact with a second ori.

Front 183

DNA replication in Prokaryotes

Back 183

The DnaG primase and Dna B helicase complex binds to the lagging strand.
Periodically the complex pauses to allow DnaG primase to synthesize an RNA primer for the lagging strand while the DnaB helicase unzips the strand. Following the complex ss binding proteins bind to DNA to keep it open.
The strand is elongated onto a previous Okazaki fragment (*1000-2000 bases*) by DNA Polymerase III. Elongation stops when it runs into another RNA primer. DNA polymerase III dissociates and DNA polymerase I binds to remove the RNA primer and fill in the gap with DNA (called nick translation).
Following the filling of the gap DNA Polymerase I dissociates and the nick is sealed by DNA ligase. During this process extra twists are being added to the dna which is eleviated by DNA gyrase a four subunit protein. The gyrA subunits catalyze transesterfercation reactions, while gyrB subunits cautalyze hydrolysis of ATP to mediate strand passage through the broken strands

Front 184

DNA replication in Eukaryotes

Back 184

In the eukaryotic leading strand the clamp loading factor RFC helps to load the sliding clamp (PCNA), which helps to initiate the binding of DNA polymerase δ.
In the eukaryotic lagging strand Replication protein A (RPA)(*as compared to ss binding protein in prokaryotes*) binds single stranded DNa once parental strands separate. DNA polymerase a is complexed with primase and they bind to the opened DNA strands and lay down primer.
oThe complex synthesizes a 10 bp RNA primer and then elongates it with 15-30 DNA’s. the complex then dissociates and DNA polymerase comes in to extend the okazaki fragment. (*130-200bp*) RNaseH degrades the RNA primer but leaves one behind and FEN1 removes the last nucleotide. DNA polymerase δ then fills the gap and DNA ligase fills the nick

Front 185

What are transposons?

Back 185

mobile segments of DNA that move an essentially random manner from one site to another

Front 186

What is DNA polymorphism?

Back 186

inherited differeneces in dna base sequences---some associated with disease

Front 187

What is a clone?

Back 187

the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vivo.To "clone a gene," a DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymes and then united with a plasmid that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector in the lab, it is called a "recombinant DNA molecule." Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the host cell DNA

Front 188

What is recombinant DNA?

Back 188

form of DNA that does not exist naturally, which is created by combining DNA sequences that would not normally occur together

Front 189

What does restriction fragment length polymorphism refer to

Back 189

Point mutations can occur in a recognition site for a restriction endonuclease and short DNA fragments are created (restriction fragments)

Front 190

How can restricition fragments be used to identify disease?

Back 190

Individuals with mutations will have variations in the length of restriction fragments produced compared to normal individuals, and the abnormal strands are called RFLP

Front 191

What is DNA polymorphism?

Back 191

2 or more allelic forms within a polymorphisms that can arise from point mutations, insertions, and deletions; some can be associated with disease and can be used as markers for the disease

Front 192

What is a clone?

Back 192

he procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vivo

Front 193

What is recombinant DNA?

Back 193

form of DNA that does not exist naturally, which is created by combining DNA sequences that would not normally occur together

Front 194

What does restriction fragment length polymorphism refer to

Back 194

Point mutations can occur in a recognition site for a restriction endonuclease and short DNA fragments are created (restriction fragments)

Front 195

How can restricition fragments be used to identify disease?

Back 195

Individuals with mutations will have variations in the length of restriction fragments produced compared to normal individuals, and the abnormal strands are called RFLP

Front 196

What does reverse transcriptase do?

Back 196

reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse transcribed into a single strand cDNA. Normal transcription involves the synthesis of RNA from DNA; hence, reverse transcription is the reverse of this.

Front 197

What is DNA polymorphism?

Back 197

more allelic forms within a polymorphisms that can arise from point mutation

Front 198

What is cloning?

Back 198

process of making multiple copies of a defined DNA sequence. Cloning is frequently used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production.

Front 199

What is recombinant DNA?

Back 199

form of DNA that does not exist naturally, which is created by combining DNA sequences that would not normally occur together.[1] In terms of genetic modification, recombinant DNA (rDNA) is introduced through the addition of relevant DNA into an existing organismal DNA, such as the plasmids of bacteria, to code for or alter different traits for a specific purpose, such as antibiotic resistance.[1] It differs from genetic recombination, in that it does not occur through processes within the cell, but is engineered.

Front 200

What does restriction fragment length polymorphism refer to?

Back 200

Point mutations can occur in a recognition site for a restriction endonuclease and short DNA fragments are created (restriction fragments)

Front 201

How can RFLP's be used to identify diseases?

Back 201

Individuals with mutations will have variations in the length of restriction fragments produced compared to normal individuals, and the abnormal strands are called RFLP

Front 202

Know what restriction endonucleases are

Back 202

Restriction endonucleases cleave only at restriction sites (recognition sites) and create fragments of DNA
i. They recognize short (4-6bp) called palindromes(sequence is the same on both strands, not complementary, but the same) and unique
ii. Some have sticky ends, some have blunt ends
iii. 900 found
iv. First letter in name is the genus, next 2 are for species, the next for the strain, and the number for what order they were identified in

Front 203

What are steps is Southern Blotting?

Back 203

1. dna extracted form white cells
2. dna cleaved with restriciton enzyme-people can have different restriciton enzymes
3. agragose gel electrophoresis-fragments separated by size
4. denature dna into single strands and transfer(by blotting action) to nitrocellulose membrane, creating replica of the gel
5. hybridization w/32
6. expose xray film P-labeled probe
dna from 2 individuals differ in region recogmnized by probe.

Front 204

Understand basics of PCR

Back 204

PCR – polymerase chain reaction
i. Need primers, dNTPs, and a thermostable DNA polymerase (Taq) and your sample or template DNA
1. Primers are short sequences designed to be complementary to the sequence you want to copy, and 2 are needed for each sequence (one for 3’ end of sequence on each strand)
ii. The mix of necessary items is placed in a thermal cycler and several steps take place:
1. Denaturation: ~94 degrees C, causes the DNA double strands to split apart
2. Annealing: mixture is cooled (Tm – 5 degrees) and the primers bind to the template strand
3. Elongation: ~72 degrees and the primers are extended using the dNTPs
4. Several cycles occur, and 2 times as much DNA is created each time
iii. Primer set that is specific to the mutation is used for diagnosis, there is no amplification if there is no mutation – dark band on an electrophoretic gel for mutation
iv. Used for DNA fingerprinting as well by amplifying VNTR (variable number or tandem repeat sequences) of the loci

Front 205

Let's make some DNA

Back 205

use mRNA to make a DNA complementary to an mRNA using reverse transcription
i. Isolate mRNA in the cell using the polyA tail specific to mRNA
ii. Reverse transcriptase is added to mRNA with dNTPs and primers
iii. Primer anneals and reverse transcriptase makes a cDNA copy that makes a hook at the end, then an alkaline base removes RNA and DNA polymerase extends hook into complementary strand
iv. S1 will cleave the hook

Front 206

Know how antibiotic resistance works for selecting bacterial transformants

Back 206

look up

Front 207

Know why plasmid vectors often have antibiotic resistance genes associated with them.

Back 207

bacteria are grown in presence of antibiotics thus selecting for cells containing the hybrid plasmids which provide antibiotic resistance.