BIO112 Exam 2

replication fork

tear in the DNA where it’s separating during replication

replication origin

specific sequence of nucleotides that is recognized

does the replication origin have more AT or GC pairs?

ATs

leading strand synthesis

the strand being made 5’ to 3’ in the same direction that its fork is moving

lagging strand synthesis

the strand being made 5’ to 3’ in the opposite direction that its fork is moving

Okazaki fragments

short 5’ to 3’ sections of DNA which the lagging strand is discontinuously synthesized in

how fast does replication happen

• very fast

• eukaryotes: 50 nucleotides/sec

• bacteria: 750 nucleotides/sec

bubble

place where strands are separated at the origin of replication

how many origins of replication do bacteria have

just one

how many origins of replication do eukaryotes have

multiple. bubbles keep growing until they run into each other

what species is replication most studied in?

e coli

oriC

origin of replication in chromosomal e coli dna

three significant oriC sequences

1. AT-rich region

2. dnaA boxes

3. GATC methylation sights

dnaA boxes

specific sequence that protein binds to within oriC

boxes

published DNA sequence

GATC methylation sites

control if replication is turned on/off

DNA helicase

separates the 2 DNA strands

supercoiling

knots formed from overtwisting

DNA gyrase/ topoisomerase ii

travels ahead of helicase and alleviates supercoiling

SSBPs (singe-strand binding proteins)

bind to the separated strands to keep them apart

(DNA) primase

synthesizes short RNA primers which start DNA synthesis

what type of enzyme is DNA primase?

an RNA polymerase

is a primer made by primase needed on the leading strand

yes

why is an RNA primer necessary?

because DNA polymerase can’t start synthesis of a polynucleotide, it can only add nucleotides to the 3’ end

DNA polymerase III

catalyzes elongation of new DNA @ replication fork

how many phosphates do nucleotides have before being added to strand?

3

what are the 2 leftover phosphates called?

inorganic phosphate / pyrophosphate / PPi

sliding clamp

subunit of DNA pol III that clamps DNA to enzyme and slides

DNA ligase

makes last phosphodiester bond between okazaki fragments

DNA pol I

removes the previous primer and fills in the gap with DNA

5’ —> 3’ exonuclease activity

chews away at the ends of RNA primer

DNA pol II

polymerase that repairs DNA

what causes thymine dimers?

uv radiation

thymine dimers

inappropriate bond between two thymines next to each other which prevents replication from taking place

how is a thymine dimer fixed?

nuclease cuts it out, DNA pol II will fill in the gap, and ligase will seal it

why does a problem occur at the 3’ end of the chromosome during replication in eukaryotes?

• Eukaryote DNA is linear

• DNA pol can’t initiate DNA synthesis

• DNA pol can only move 5’—>3’

• There is no place to add a primer at the 3’ end

telomeres

DNA sequences at the end of chromosomes

composition of telemorase

part RNA, part protein

telemorase

adds a species-dependent telomere repeat sequence to the 3' end of telomeres

what is the significance of the RNA in telemorase?

• is complementary to the DNA sequence found in the telomeric repeat

• allows telomerase to bind to the 3’ overhang

what are potential benefits and harms of stimulating telemerase?

Benefits: prevent DNA damage and aging

Harms: could cause cancer

what cycle do telomeres do?

binding-polymerization-translocation

do bacteria have telomeres

no

do telomeres prevent the shortening of DNA

no, but they do postpone the erosion of important genes near the ends of DNA

what does it mean that DNA pol III is a processive enzyme

it remains attached to the template as it is synthesizing the daughter strand

what is the alternate name for the clamp?

β subunit

what happens without the β subunit

DNA pol III falls off the template after a few dozen nucleotides, slowing down replication to 20 nucleotides per second

what happens with β subunit?

DNA pol III stays on the template long enough to make 50,000 nucleotides at a rate of 750/second

DNA has a _____ degree of fidelity

high

3 reasons for high fidelity

1. instability of mismatched pairs

2. Configuration of the DNA polymerase active site

3. Proofreading function of DNA polymerase

Proofreading function of DNA polymerase

• DNA pol can identify a mismatch nucleotide and remove it from the daughter strand

• enzyme uses 3’ to 5’ exonuclease activity to remove the incorrect nucleotide

when/where does replication end

• when oppositely advancing forks meet

• at T1 or T2

what is the final step of DNA replication

DNA ligase covalently links all 4 DNA strands

Catenanes

2 intertwined molecules which result from replication of circular DNA

topoisomerase II enzymes

separate catenanes by cutting the circular DNA rings, allowing them to separate, and seals the cut

transcriptional units

alternate name for genes

is mRNA processed in bacteria?

no

different functions of DNA sequences

1. code for proteins

2. bind to proteins which carry out a function on DNA

some functions of noncoding genes

• promoters

• regulatory sequences

• terminators

regulatory sequences

site for the bonding of regulatory proteins which influence gene expression

what does the terminator do?

disrupt production of RNA and end transcription

is the promoter transcribed

no

RNA Polymerase

• synthesizes RNA

• no need for primers / primase

• can only lengthen 5’ —> 3’

Steps of Transcription

1. initiation

2. elongation

3. termination

what happens in initiation?

• promoter allows RNA pol to recognize where gene starts

• formation of closed and open complexes

closed promoter complex

• when RNA first binds to promoter and DNA is still closed

• not very strong, RNA pol can easily come off

open promoter complex

• tight, high affinity binding, once the DNA is unwound

• transcription bubble is formed

transcription goes in ____ direction(s)

1

transcription factors

transcription regulator proteins that mediate the binding of RNA pol and initiation of transcription

which RNA polymerase does transcription?

RNA pol II

+1 site

first nucleotide in a gene that is used as a pattern

what number nucleotide is to the left of the +1 site

-1

upstream

nucleotides in the opposite direction of transcription

downstream

further in the direction of transcription

Pribnow box

TATAAT sequence at -10 position in prokaryotes

consensus sequence

frequent sequence in nature

-35 sequence

TTGACA sequence at -35 position

what sequences does RNA pol recognize to bind to dna in prokaryotes?

-10 and -35

does the sequence of nucleotides between the -10 and -35 sequences matter?

No, but it has to be the correct length

template strand

the strand which is used as a template for the RNA

coding strand

DNA strand whose base sequence is identical to the base sequence of the synthesized RNA

What is the solution to a cell needing lots of one RNA

a gene can be transcribed simultaneously by several RNA polymerases

Where are the 5’ ends in christmas tree transcription

the tips of the “branches”

structural gene

• encodes a polypeptide

• is transcribed to mRNA

nonstructural genes

• do not encode a polypeptide

• are transcribed into tRNA or rRNA (not translated)

where is the TATA box in Eukaryotic genes

-25

basal transcription

low level of transcription from just core promoter

core promoter

• short promoter that only consists of the TATA box

GC and CAAT boxes

regulatory elements in eukaryotic cells that affect the binding of RNA pol to the promoter

enhancers

regulatory elements that stimulate transcription

silencers

regulatory elements that inhibit transcription

where are GC and CAAT boxes located

• exact location varies

• usually around -50 to -100 region

what are the two types of elements that control gene expression

• cis-acting elements

• trans-acting elements

cis-acting elements

• dna sequences that exert their effect on nearby genes. (located on the same dna molecule)

• Ex: TATA box, enhance, silencer

trans-acting elements

regulatory proteins that bind to cis-acting element DNA

where does RNA splicing taking place

• only in eukaryotes

• the nucleus

colinearity

the coding strand corresponds to the sequence of amino acids in the polypeptide

are mRNA and coding strand always colinear

no in eukaryotes

pre-mRNA/hnRNA/primary transcript

RNA while it is still in the nucleus and hasn’t been spliced