Quiz 2

• synthesizes short RNA molecule
• reads DNA template and adds nucleotides in order to generate RNA primer

• synthesizes a new strand of DNA
• adds nucleotides to the 3' end of the new strand

• separates double helix at replication fork
• denatures template in front of replication fork

• primer for DNA synthesis
• Enzyme that joins two molecules of DNA together
• closes the gap after repair, telomere extension or DNA synthesis

• Recognize the damage with special proteins
• Remove the damage with nucleases
• Fill in the missing DNA with DNA polymerase
• Close the gap DNA ligase

The direction of DNA synthesis is 5'-3', so DNA synthesis must be discontinuous on the lagging strand in order to synthesize both strands of DNA at in the same direction

DNA synthesis occurs during the S phase of the cell, which usually lasts about 8 hours.  A human diploid cell contains 46 chromosomes which make up the human genome. At the end of S phase, each chromosome has been replicated to produce two complete copies.

• damage due to “thermal collisions” and oxidative damage Depurination and deamination; Altered bases
• Mismatch: Wrong base incorporated during replication (A not matched to G)
• DNA mismatch repair system: Group of proteins that recognize and repair mismatchs

• damage due to exposure to chemicals: DNA adducts
• Radiation: UV dimers; double strand breaks
• Nucleotide excision repair (NER): DNA adducts – bulky adducts added to the DNA

• 1.  Initiation proteins bind at the replication origins. Denature the DNA
• 2.  Helicase unwinds the double helix at the origin of replication
• 3.  Single strand binding proteins bind and keep the strands apart
• 4.  Primase forms a short 10 base RNA primer at the FORK on both strands
• 5.  DNA polymerase adds bases to the 3’ end of the primers

(2) Helicase unwinds the double helix at the origin of replication

(3) Single strand binding proteins bind and keep the strands apart

(4) Primase forms a short 10 base RNA primer at the FORK on both strands

(5) DNA polymerase adds bases to the 3’ end of the primers

• Telomerase binds to the template of the lagging strand at the end of the chromosome to facilitate the addition of telomeres.
• Telomerase has a bound RNA template that adds telomeres to the ends of chromosomes  via RNA templated DNA synthesis.
• The lagging strand is then completed by DNA polymerase via DNA templated DNA synthesis

keeps the double strands apart until polymerase passes

• takes the kinks out of the DNA and rotates the strands ahead of the replication fork
• removes kinks and tensions in front of replication fork

10 base pair sequence formed by primase at replication fork during DNA synthesis

• Particular sequence of DNA  
• Recognized by initiator proteins  
• Causes DNA to melt and become denatured
• Hydrogen bonds are broken and strands separated
• Where DNA synthesis begins

Y shape where the two DNA strands split apart during replication; come in pairs

G/T rich sequences on ends of chromosomes -GGGGTTA-

Enzyme which adds DNA to ends of chromosomes (onto telomeres)

DNA polymerase can only add onto the 3' end of an -OH group and thus requires a primer to provide an -OH group on which to add the first nucleotide

between phosphate of 5'C of one nucleotide and -OH group of 3'C of other sugar

Phosphodiester Bond

Adds one nucleotide at a time  Adds to 3’ end of growing chain  Grows in 5’ to 3’ direction  Forms a phosphodiester bond  Reads the parental strand

• incoming nucleotide pairs with a base in the template strand
• DNA polymerase catalyzes covalent linkage of nucleotide into growing nucleotide strand

keeps DNA polymerase on the DNA

• DNA polymerase
• primase
• DNA ligase
• Sliding clamp protein
• helicase
• topoisomerase
• single strand binding proteins

Wrong base incorporated during replication

• Leads to a change (mutation) in the DNA sequence
• depurination: Loss of A and G; Spontaneous
• deanimation of cytosine: spontaneous

checks each nucleotide after added Removes wrong nucleotide and re-adds 3’-5’ exonuclease activity 1 error every 107 nucleotides

Group of proteins that recognize and repair mismatch

• after replication deletion of base pair
• loss of purine A
• T not paired with A
• after replication, A-T nucelotide pair deleted on one strand, but replicated correctly on the other

• deanimation of cytosine (C)
• C turns to U
• U pairs with G
• after replication, one strand is made correctly, with G pairing with C, and the other incorrectly with U pairing with A

two thymines become one thymine dimer

• modified bases or altered bases
• U in the DNA
• 8-oxo G
• Methylated A
• repairs deanimation

• DNA adducts – bulky adducts added to the DNA
• DNA repair systems for environmental damage and spontaneous changes mechanism

• Single stranded
• U-A pairings possible
• internal base pairing – leads to specific 3D RNA Structures

• Promoter and Gene regulatory region
• Start of transcription
• Downstream
• Upstream

sequences involved in initiation of transcription

sequences involved in regulating expression of the gene.

coding and noncoding sequences that will appear in the mature, processed mRNA

intervening sequences which are removed from primarytranscription; do not contain coding sequences

sequences (AAUAA) in which determines where polya tail will be added

sequence at which RNA polymerase stops transcribing

noncoding sequence contained in exon I, which comes before the start of translation

noncoding region contained in the final exon, which comes after the stop codon

start of translation; 1st codon of the coding region; codes for methionine

stop codon for translation - stops translation of the mature mRNA

transcribes most rRNA genes

all protein coding genes, miRNA genes, plus genes for other non-coding RNA (those in spliceosomes)

tRNA, 5S rRNA, genes for many other small RNAs

• 1. TFIID binds at TATA (via TBP –TATA binding protein)
• 2. TFIIB’s binds to TFIID
• 3. Binding of RNA polymeraseTFIIF, TFIIE, TFIIH
• 4. Phosphorylation of RNA Polymerase and release of TF’s

Proteins that bind to specific sequences in the promoter of the gene

• Contains control sequences for transcription
• Binding site for RNA polymerase (Promoter)
• Contains site of initiation (Promoter)

Where first nucleotide comes in and transcription begins Referred to as +1

Towards the coding region of the gene

Away from coding region of the gene

• 1.  Formation of 5’ cap
• 2.  Addition of 3’ poly (A) tail
• 3.  Removal of introns and joining of exons Splicing

• Three bases of 5’ end are termed the 5’ cap
• 1st of the 3 bases is added after transcription; guanine is added in opposite direction to last nucleotide on the pre-mRNA; 5’-5’ linkage
• Important for translation

• Cut 10 to 20Nucleotides pass site on primary transcript
• first nucleotide: +1sequence that tells us where to put polyA

• Length varies from 3 to 200 nucleotides
• appears to be important for stability or half-life of mRNA; shorter tails - shorter half-life; longer tails - longer half-life
• Tail gets shorter as mRNA ages Proteins associate with the poly (A) tail