gene 3200: unit 3 exam

why are different genes expressed in different places?

even though the chromosomes and genes in each cell should be the same, different tissues and cell types transcribe different genes

what are genes?

sequences of DNA that are transcribed into RNA

includes the transcribed sequences as well as the sequences that control transcription

is all DNA transcribed into RNA?

no

is all RNA translated into protein?

no

what are the 5 important characteristics of genetic material?

1. replication (must be able to make exact copies)

2. stability (structural and chemical integrity)

3. mutability (potential for mutations for genetic variability)

4. expression (encodes phenotype)

5. heritability (passed from parent to offspring)

what are the repeating units of DNA and RNA? (3)

= nucleotides

1. pentose sugar

2. phosphate group

3. nitrogenous base

what sets RNA and DNA apart?

RNA has an extra oxygen, making DNA much more stable than RNA

what makes up the phosphodiester backbone of DNA and RNA?

pentose sugars and phosphate groups

what are the purines and their basic shape?

adenine and guanine

2 ringed nitrogenous base

what are the pyrimidines and their basic shape?

cytosine, thymine, uracil

1 ringed nitrogenous base

what nitrogenous bases go together?

A : T = 1

G : C = 1

A + G = T + C

what does it mean to be antiparallel?

the orientation of the two strands of DNA run parallel to each other but in opposite directions

in DNA, one strand runs in the 5' to 3' direction while the other runs 3' to 5'

where is the phosphodiester bond?

bond between the 5’-PO4 and 3’-OH groups of adjoining nucleotides on each strand

where is the hydrogen bond?

bond between complementary bases of opposite strands (intermolecular)

how many H bonds are between A-T base pairs?

2

how many H bonds are between G-C base pairs?

3

is more energy required to denature G-C pairs or A-T pairs?

G-C since its 3 H bonds

what is a hairpin in DNA?

a specific structure formed when a single strand of DNA folds back upon itself, creating a loop which is stabilized by base pairing within the same strand

what is a stem in DNA?

a double-stranded region formed by intramolecular base pairing within a single-stranded nucleic acid molecule

how do prokaryotic vs eukaryotic genetic material differ?

1. location:

   1. prokaryotic is found in cytoplasm

   2. eukaryotic is found in the nucleus

2. nucleus: prokaryotic don’t have nuclei

3. structure:

   1. prokaryotic is a single circular chromosome

   2. eukaryotic is multiple linear chromosomes

4. packaging:

   1. prokaryotic aren’t wrapped in histones

   2. eukaryotic are wrapped in histones (chromatin)

5. replication + transcription:

   1. prokaryotic have transcription and translation occurring simultaneously

   2. eukaryotic have transcription in the nucleus and translation in the cytoplasm

what is supercoiled DNA?

additional winding/twisting of double-helix DNA, allowing more information to fit into a small space

what is positive supercoiling?

clockwise direction of winding, overrotated

what is negative supercoiling?

counterclockwise direction of winding, underrotated

is most DNA positively or negatively supercoiled?

negatively for easier separation of strands for replication and transcription

what are topisomerases?

add or remove rotations in DNA by breaking and then rejoining DNA strands

what is chromatin?

DNA and associated proteins of a chromosome

protein component is essential for condensation, segregation, and organization of chromosomes

what are histones?

small, positively-charged, highly conserved proteins found in all eukaryotes to bind to and neutralize negatively charged DNA

what is the composition of a chromosome?

about ½ DNA, ½ protein (1/2 of this is histone proteins)

what are the 5 types of histone proteins?

H1, H2A, H2B, H3, H4

what is euchromatin?

relaxed chromatin, usually transcriptionally active

lightly stained regions of chromosomes

what is constitutive heterochromatin?

tightly condensed, usually transcriptionally inactive (silenced)

darkly stained regions of chromosomes

highly compacted even during interphase

what is facultative heterochromatin?

condensed or relaxed under certain conditions

what is a restriction enzyme?

recognizes a specific sequence of bases anywhere within the DNA
a protein produced by bacteria that cuts DNA at specific sequences, creating fragments with predictable ends

what are recognition sites?

place where restriction enzyme cuts

each enzyme cuts at the same place relative to its specific recognition sequence

what are cohesive ends?

5’ and 3’ overhangs

what are blunt ends?

no overhang

what is gel electrophoresis?

different types of gels can be used to separate macromolecules based on size; generates resolving fragments

what factors can affect the migration or final position of a macromolecule after gel electrophoresis?

1. size

2. molecular charge

3. molecular shape

what is a southern blot?

membrane that contains transferred DNA

what is a northern blot?

membrane that contains transferred RNA

what is a western blot?

membrane that contains transferred proteins

when do errors in DNA occur?

whenever information is copied—the more times it is copied, the greater the potential number of errors

why is DNA polymerase so accurate?

it has a proofreading mechanism (3’ → 5’ exonuclease)

what were the 3 proposed mechanisms for DNA replication?

1. semiconservative replication

2. conservative replication

3. dispersive replication

what is the semiconservative model of DNA replication?

each daughter duplex contains one parental and one daughter strand

what is the conservative model of DNA replication?

one daughter duplex contains both parental strands and the other contains both daughter strants

what is the dispersive model of DNA replication?

each daughter duplex contains interspersed parental and daughter segments

what was the meselson-stahl experiment?

confirmed DNA was semiconservative

• grew e. coli with a heavy and a light nitrogen isotope

• in the first generation, DNA only showed one intermediate band and not 2 light and heavy bands, ruling out the conservative model

• in the second generation, DNA showed both intermediate and light bands, ruling out dispersive model

where does theta replication occur?

in E. coli and other bacteria

• single origin of replication

• bidirectional replication from the origin

where does linear replication occur?

in all eukaryotes

where is replication initiated?

at origins (Ori) and is initiated with an RNA primer

how does DNA polymerase execute replication?

is template driven through H-bonding, occurs in the 5’ → 3’ direction, and requires free 3’-OH

catalyzes the formation of phosphodiester bonds with the 3’-OH of the new strand and the 5’-PO4 of the next nucleotide

part of the strand is synthesized continuously while other parts of the same strand are synthesized semi-discontinously

what are the 4 stages of DNA replication in E. coli?

1. initiation: OriC

2. unwinding: single strand binding proteins, helicase, DNA gyrase

3. elongation: primase, RNA primer, DNA pol III, leading strand, lagging strand, okazaki fragments, DNA pol I, ligase

4. termination

what happens in the initiation phase of E. coli DNA replication?

an initiator protein binds to the a single replication origin and causes a short section of DNA to unwind

what happens in the unwinding phase of E. coli DNA replication?

helicase binds to the lagging strand and moves in the 5’ → 3’ direction to break H bonds, single stranded binding protein stabilize single stranded regions, DNA gyrase relieves torsional strain in front of each fork

what happens in the elongation phase of E. coli DNA replication?

DNA synthesis is initiated at RNA primers and synthesized by primase

a new primer is needed for each leading strand and each okazaki fragment

what are the characteristics of RNA primers?

complementary and antiparallel to each template strand

what is the leading strand?

synthesis of one daughter strand occurs continuously in the same direction as the replication fork progresses

what is the lagging strand?

synthesis of the daughter strand occurs discontinuously, in the opposing direction to fork progression, via short segments called okazaki fragments

what does DNA polymerase I do?

removes and replaces primers

completes replication by using:

1. 5’ to 3’ exonuclease to remove the RNA primers

2. 5’ to 3’ polymerase activity to add DNA nucleotides to the 3’ end of the DNA segment preceding the primer

what does DNA ligase do?

catalyzes formation of a phosphodiester bond between adjacent DNA segments (3’-OH and 5’-PO4)

what does DNA polymerase III do?

elongates DNA

what is the origin-recognition complex (ORC)?

binds to origins to initiate DNA replication during G1 of interphase (replication occurs only during S phase)

what are telomeres?

consist of specific repetitive DNA sequences and don’t contain genes in order to maintain integrity of chromosomal ends and protect the ends of chromosomes to prevent chromosome fusion

what is the end-replication problem?

in linear chromosomes, the lagging strand cannot fully replicate its 5’ end after primer removal, causing gradual shortening of chromosomes after each replication cycle

alleviated by telomeres and telomerases

• telomeres contain a 3’ G-rich overhang that folds over and forms a t-loop (a lasso-like structure formed at the ends of chromosomes) by H-bonding with the complementary strand

• a reverse transcriptase of telomerase (TERT) copies the DNA template on htR to add DNA repeats onto the telomere end

  • hTR is the RNA component of telomerase that contains the sequence that is complementary to the tandem repeat on the telomere end

what is the shelterin complex?

6 proteins that bind the t-loop and help to:

1. mediate t-loop formation

2. protect the end sequences

3. recruit and regulate telomerase

what is the TERT gene?

provides instructions for making telomerase reverse transcriptase but also can give you cancer

what is the polymerase chain reaction (PCR)?

a lab technique used to make many copies of a specific DNA segment

what is required for PCR?

1. a dsDNA template containing the target sequence to be amplified

2. a mixture of all 4 dNTPs

3. 2 primers for opposite strands of the target DNA

4. an aqueous buffer solution

5. thermocycler

6. a thermo-stable DNA polymerase (most common is Taq polymerase, which lacks 3’ → 5’ exonuclease)

what are the steps in each PCR cycle?

1. denaturation of DNA into single strands

2. hybridization of primers to their complementary sequence

3. DNA synthesis

amount of DNA is doubled with each cycle

what are some of the limitations of PCR?

1. need prior knowledge of part of the sequence of target DNA

2. very susceptible to contamination

3. Taq polymerase lacks 3’ → 5’ exonuclease (proofreading)

4. size limitation of DNA fragments

why is RNA’s secondary structure important?

inverted repeats in RNA sequence can form intramolecular H-bonding called hairpins or stem-loops

what are some comparisons between DNA and RNA structures?

what is transcription?

the synthesis of RNA from the template strand of DNA

what are some comparisons between DNA replication and transcription?

what is the coding strand?

non-template strand → 5’ to 3’

the RNA base sequence in transcription is identical to the base sequence of the coding strand (except for U/T)

what is the non-coding strand?

template strand

antiparallel and complementary to the RNA base sequence of transcription

what is the transcription bubble?

a localized region of unwound DNA that forms during transcription, allowing RNA polymerase to access and transcribe the template strand of DNA into RNA

moves 3’ → 5’ with respect to the template DNA but the synthesis occurs 5’ → 3’

what is the promoter in transcription?

determines where transcription initiates but is NOT part of the transcribe sequence

RNA polymerase binds here and transcription starts at a defined distance from the promoter (+1)

what is the consensus sequence?

the most common base sequence found at a specific location or alignment in DNA

what is sigma factor?

the core RNA polymerase cannot bind the promoter or initiate RNA synthesis without a sigma factor

what are the 3 steps of bacterial transcription?

1. initiation

2. elongation

3. termination

what happens during initiation of bacterial transcription?

the holoenzyme starts unwinding at the -10 position of the promoter

needs the sigma factor to bind which then dissociates from the core polymerase after intiation

what happens during elongation of bacterial transcription?

transcription bubble forms, newly synthesized RNA forms complementary base pairs with template DNA

what is Rho-dependent termination?

Rho: an ATP-dependent helicase that unzips DNA by breaking H bonds

recognizes and binds to a Rut site in the RNA (C-rich region that lacks secondary structure) and moves 5’ to 3’ until it encounters RNA polymerase, which has paused near the 3’ end of transcription

what is Rho-independent termination?

RNA polymerase pauses at strings of U’s allowing the hairpins to form at the inverted repeats which causes pairing between DNA and RNA to be destabilized, leading to release of RNA

are eukaryotic or prokaryotic promoters more diverse?

eukaryotic - eukaryotic transcription is a lot more complex than prokaryotic

in eukaryotes, how many different RNA polymerases recognize different promoters and transcribe different types of RNA?

3

what are low levels of transcription initiation mediated by?

basal transcription apparatus that assembles at the core promoter

what are high levels of transcription initiation mediated by?

mediated when specific transcription factors bind to regulatory promoter sequences and/or more distant enhancers

what is the core promoter?

site for assembly of basal transcription apparatus, contains atleast one consensus sequence

RNA pol II promoter sequence

what is the TATA box?

a common consensus sequence that marks the beginning of transcription for a gene

most common sequence, at -25

RNA pol II promoter sequence

what is a regulatory promoter?

binding sites for specific transcription factors

RNA pol II promoter sequence

what are the steps of eukaryotic transcription?

1. intiation

2. elongation

3. termination

what happens in the initiation phase of eukaryotic transcription?

transcription factors and RNA polymerase II assemble at the promoter region, forming a pre-initiation complex (PIC), which then unwinds the DNA, allowing the template strand to be read and transcription to begin

• RNA pol II and general transcription factors assemble on the core promoter

• TFIID (general TF) binds to the TATA box

• specific transcription factors binds to enhancers and regulatory promoters and interact with the basal transcription apparatus through mediator and coactivator proteins

• 11-15 bp of DNA is unwound and positioned within the active site of RNA polymerase

what happens in the elongation phase of eukaryotic transcription?

RNA polymerase, after initiating transcription, moves along the DNA template strand, adding nucleotides to the growing RNA transcript in a 5' to 3' direction, forming the mRNA molecule

• RNA pol II leaves promoter and general TFs available to form another open complex with another RNA pol II

what happens in the termination phase of eukaryotic transcription?

a poly(A) signal sequence in the transcribed RNA triggers cleavage and polyadenylation, followed by the release of the RNA transcript and the RNA polymerase from the DNA template

• cleavage occurs while transcription is still taking place

• polyadenylation takes place at 3’ end of cleaved mRNA

• an exonuclease (Rat1) degrades RNA in 5’-3’ direction

• transcription terminates when Rat1 reaches RNA pol II

what are some posttranscriptional modifications to eukaryotic precursor mRNA? (3)

1. addition of 5’ cap

2. 3’ cleavage and addition of polyA tail

3. RNA splicing

what is the significance of 5’ cap?

facilitates binding of ribosome to 5’ end of mRNA, increases mRNA stability, enhances RNA splicing

what is the significance of the 3’ cleavage and addition of polyA tail?

increases stability of mRNA, aids in export of mRNA from the nucleus, and facilitates binding of ribosome to mRNA