Molecular Recombination
Exchanging genetic material between two non sister chromatids
Crossover
Complex of proteins responsible for DNA replication
Replisome
Functions of DNA polymerase
5'---3' DNA polymerase synthesizes daughter strand DNA
5'---3' exonuclease removes RNA primer and replaces it with DNA
3'---5- exonuclease recognizes and replaces mismatched basepairs
Making RNA from DNA template
Jeff
Describe 5' Capping and give the enzyme responsible and functions it provides (2).
Modifies pre-mRNA by adding a 7-methyl guanosine to the 5' end.
CDT-RNA pol 2
Protects from degradation and allows ribosome to recognize mRNA
Describe important RNA sequences on intron
5' splice site -GU-
3' splice site -AG-
Branch point -A-
Describe Self-splicing
The intron itself catalyzes its own removal (ribozyme) using 2 transesterifications.
Exchanging of genetic material between homologous chromosomes
Homologous recombination
Semi conservative replication refers to
the correct method of DNA replication
dsDNA separates into ssDNA and both strands are replicated in the 5'----3' direction
What are the important DNA sequences and proteins involved in prokaryotic initiation?
DNA sequences: AT rich region and DNAa box
Proteins: DNAa protein(initiation factor), single stranded binding proteins, helicase, gyrase
Requirements for transcription (3)
DNA template
Ribonucleotide triphosphates (tRNPs)
Transcription apparatus: RNA polymerae + many proteins
Describe 3' polyadenylation, the enzyme involved and the functions it provides (3).
The addition of 150-200 adenine on the 3' tail on pre-mRNA.
Poly A polymerase
Protects from degradation, facilitates the attachment of ribosome, exports mRNA to cytoplasm.
Describe the first step of Splicing via Spliceosome
Pre-mRNA is cut at 5' splice site between exon 1 and -GU-
Frees exon 1
Lariat structure is formed
What are RNA binding proteins?
activators/repressors that bind to RNA splice site and dictate RNA splicing.
_______ allows recombination to occur
Crossover
spontaneous isomerization from a regular amine/keto to an imino/enol form
What are the important DNA sequences and proteins involved in Eukaryotic initiation?
DNA sequences: AT rich region, 11 base pair consensus sequence
Proteins: ORC, Cdc6, Cdt1, helicase, single stranded binding proteins, gyrase
5'_______3' is called_______
3'_______5' is called_______
Coding strand (sense strand)
template strand (anti sense strand)
Define RNA Splicing and what enzyme is responsible.
Removing introns and connecting exons together.
Spliceosome
Describe the second step of splicing via spliceosome
Pre-mRNA is cut between 3' -AG- and exon 2
Frees exon 2
Lariat structure is released and degrades
Define Alternative Splicing
The same pre-mRNA can be spliced in more than one way to yield multiple mRNAs and in turn, multiple proteins
Describe Double Strand Break
Endonuclease cuts one homologous chromosome
This occurs in prophase 1
4 requirements of DNA replication
1) DNA template
2) primer
3) DNA polymerase
4) deoxynucleoside triphosphates (nuctleotides)
Describe leading strand synthesis in prokaryotes
Primase synthesizes primer which provides a free 3' OH group, DNA polymerase 3 synthesizes leading strand from leading strand template
Length of DNA transcribed into RNA
Transcription Unit
5 snRNPs + proteins
each snRNP is composed of snRNA +proteins
exon 1+exon 2 are joined together forming mature mRNA
This makes the genome more efficient
Alternative splicing
Describe Resection
nucleotides are 'cleaned' from from one 3' tail
this results in two free 3' tails
Functions of DNA and chemical makeup
important for inheritance, encodes for proteins
composed of 3 chemicals, deoxyribose, phosphates, and 4 different nitrogenous bases (CATG)
Describe lagging strand synth in Prokaryotes
Primase synthesizes primer with free 3' OH group
Polymerase 3 synthesizes okazaki fragments
5'---3' exonuclease removes RNA primer and replaces with DNA
3'---5- exonuclease recognizes and replaces mismatches base pairs
ligase connect okazaki fragments
Beta clamp holds pol on DNA
DNA sequence that allows RNA polymerase to bind
promoter sequence
Duchenne Muscular Dystrophy (DMD)
What is the chemical reaction involved in Splicing?
Transesterification
Two types of mRNAs produced.
Normal 1: Immunoglobin secreted into blood
5-splice site between exons 6&7
3' splice site after exon 8
mRNA contains 123456A6B
Normal 2: Immunoglobin anchored to cell membrane
5' splice site between 6A&6B
3' splice site before 7
mRNA contains 123456A78
Single stranded binding protein binds to 3' tails
3' tail invades non sister chromatid
replaced strand becomes single strand
Formation of d loop (paired nonsiter chromatids and unpaired replaced strand)
duplication of DNA defines
DNA replication
Describe leading stand synth in Eukaryotes
Polymerase alpha synthesizes primase which synthesizes RNA primer with free 3' OH group
Polymerase Epsilon synthesizes leading strand from leading template strand continuously.
Beta clamp hold Pol onto DNA
How many promoter sequences in prokaryotes and what are their consensus sequences?
What is the spacing between each promoter region?
2
-10 region -TATAAT-
-35 region -TTGACAT-
16-19 nucleotides apart
What are the four functions of introns?
1: 5' and 3' splice sites for gene splicing
2: Alternative splicing lets more than one protein be made from a single mRNA
3: RNA itself catalyzes splicing (Ribozymes)
4: Trans splicing allows new proteins to be formed
Define transesterification
Exchanging an ester for an alcohol (hydroxal -OH)
Describe multiple 3' cleavage site, and the enzyme involved
Refers to cleavage sites within the EXON
Cleavage site 1= mRNA1=Protein 1
Cleavage site 2=mRNA2=Protein 2
Describe the formation of the double Holliday junctions
3' tail from the left basepairs with the nonsister chromatid on the right
3' tail from the right joins with nonsister chromatid on leftReplaced strand basepairs with nonsister chromatid
Formation of two Holliday junctions occur
Primary structure of DNA
chain of nucleotides help together by phosphodiester bonds
Describe lagging strand synth in Eukaryotes
DNA pol alpha synthesizes primase which synthesizes a free 3' OH RNA primer
DNA pol delta synthesizes okazaki fragments
5'---3' exonuclease removes RNA primer and replaces it with DNA
3'---5' exonuclease proofreads
DNA ligase joins okazaki fragments together
Beta clamp holds pol on DNA
3
TATA box -TATA-
CAT box -GGCCAATCT-
enhancer region further upstream
Involves inverted repeats, 6 or more Adenines, NUSA protein, RNA polymerase, stem loop structure
Rho independent termination
How many transesterifications occur?
Describe each chemical reaction.
What is the product?
2
T#1: Hydroxal on branch point -A- attacks ester attached to -G- on 5' splice site(Exon 1), groups switch.
T#2: Hydroxal on Exon 1 attacks -G- on 3' splice site, groups switch.
Exon 1+Exon 2 join together and lariat is released.
Trans splicing refers to
Describe Branch migration
Branch migration and heteroduplex is formed, basepairs are exchanged on homologous chromosomes as Holliday junctions move up and down DNA
Secondary structure of DNA and how its held together
Double helix composed of
backbone: deoxyribose and phosphate
base pairs: (G-C) (A-T)
held together by H bonds between nitrogenous bases and stacking interaction: non covalent interactions between adjacent rings
How is replication terminated in Prokaryotes?
-Replication forks meet (circular DNA)
-Tus protein binds to Ters (termination site) and blocks helicase from unwinding DNA
This complex is responsible for binding to the promoter region, initiating transcription, elongating RNA chain, terminating transcription, and is responzive to regulatory proteins
RNA polymerase
GTFs
general transcription factors that bind to TATA box and assists the binding of RNA polymerase
Describe Resolution
Heteroduplex can be cut in four possible ways
H+H and V+V yield parental type
H+V and V+H yield recombination type
Chromatin
Tertiary structure of Eukaryotes
Eukaryotic replication termination involves replicating ________
The enzyme responsible for this is _______
Telomeres
Telomerase
RNA polymerase has __ subunits.
What are their functions?
5
2alpha: interacts with regulatory proteins
1beta: binds rNTPs
1beta': binds to DNA
1sigma: binds to promoter region
Transcription Activator proteins
Recombination creates ________
genetic variation
Tertiary structure of Prokaryotes
twisted loops
Telomerase is special because
Holoenzyme binds to the promoter region and initiates transcription, what subunits is it composed of?
2alpha, 1beta, 1beta', 1sigma
What proteins and enzymes are involved in Eukaryotic transcription initiation
TF2D: binds to TATA box
GTFs: binds to additional consensus sequences
TF2D+GTFs recruit RNA polymerase
_________ refers to the hopping of telomerase RNA from 1st TTG to 2nd
Translocation
______________ are involved in studying the proteins responsible for replication
How many types of RNA polymerases do Eukaryotes have and what are their functions?
1: synthesizes rRNA
2: synthesizes mRNA, some miRNAs, some snRNA
3:synthesizes tRNAs
What process involves forming a pre-initiation complex, a conformational change with DNA and RNA polymerase, and the formation of the transcription bubble?
Eukaryotic transciption initiation
How many nucleotides are added to the 3' overhang strand during telomeric replication
~15 nucleotides
Define a nucleosome and provide the mechanism in which they are formed
nucleosome: composed of DNA (-) wrapped around histones (+) and allow DNA to form tertiary structure
CAF-1 binds to free floating histones in nucleus
CAF-1 binds to PCNA on DNA and bring histones close to DNA
DNA wraps around histones and nucleosome is formed
2alpha, 1beta, 1beta' are the subunits that make up this enzyme
core enzyme
1) RNA polymerase maintains transcription bubble
2) dsDNA unwinds to ssDNA
3) RNA polymerase synthesizes RNA using template strand
4)ssDNA winds back to dsDNA
Describe the process of replicating the complimentary strand
DNA pol alpha makes primase which provides a free 3' OH primer
DNA pol delta synthesizes okazaki fragment
ligase joins fragments together
exonuclease cleaves RNA primer
beta clamp hold DNA pol to DNA
Problem replicating telomeres?
telomere on lagging strand is shortened due to exonuclease removing RNA primer, there's no free 3' OH group
Describe transcription initiation is prokaryotes
1) sigma factor binds to core enzyme creating holoenzyme
2)sigma factor binds to -10 and -35 consensus sequences
3) holoenzyme binds tightly to DNA and beings unwinding dsDNA to ssDNA
4) holoenzyme pairs first rNTP with base of DNA and first nucleotide of RNA is transcribed from DNA to RNA
5) sigma factor is released, transcription initiates
Important consensus sequences in Eukaryotic termination
-AATAAA- and -ATTAAA-
___________ protects from DNA degradation and recombination
Telomere cap
Important DNA and RNA sequences involved in telomere replication
3' overhang strand -TTGGGG-
RNA -AACCCCAAC-
RNA core enyme undergoes transformational change and escapes promoter region, core enzyme unwinds dsDNA to single strand DNA, rNTPS are baspaired with DNA, ssDNA is rewound into dsDNA is this process
Ribonuclease function in termination in Eukaryotes?
recognizes RNA sequence and cleaves 20 basepairs downstream
Symptom of this disease is premature aging
What genes responsible for proteins that make up telomere cap could be mutated that causes this disease?
Werner syndrome
WRN, TRF1, TRF2
Rho dependent and Rho Independent
What enzyme adds A to 3' end of pre-mRNA?