On the lagging strand, DNA is made in fragments !

*!

! On the leading strand, only one initial primer is needed.! ! On the lagging strand, many primers are needed.! ! ! The RNA primers are removed by a nuclease that recognizes the RNA/DNA heterodimer.! ! A DNA repair polymerase with proofreading then lls in the gap (end of Okazaki is primer).! ! The completed fragments are nally joined/sealed by DNA ligase. !

DNA replication requires the coordination of many proteins to form the replication machine !

*!

*!

For Lagging Strand!

{!

1.! Need to unzip DNA-helicase (uses ATP)! 2.! DNA polymerase! 3.! Sliding clamp-keeps DNA pol on DNA. Putting this on requires another protein-the clamp loader (uses ATP).! 4.! Need to stabilize ssDNA so it doesnt rehybridize and keep it elongated-singlestrand binding protein (SSBPs)! 5.! Primase, a nuclease (not shown here), DNA repair pol, DNA ligase!

DNA replication requires the coordination of many proteins to form the replication machine !

DNA replication requires the coordination of many proteins to form the replication machine !

DNA replication requires the coordination of many proteins to form the replication machine !

DNA replication requires the coordination of many proteins to form the replication machine !

DNA replication requires the coordination of many proteins to form the replication machine !

*!

Telomerase replicates the ends of eukaryotic chromosomes !

*!

! Problem: DNA polymerase cannot synthesize DNA in the 3to-5 direction. And, at the ends of chromosomes there is no place to lay down an RNA primer. How are telomeres replicated?! ! Solution: Eukaryotes have special repetitive DNA sequences in their telomeres that recruit telomerase.! ! Telomerase is part protein and part RNA. It recognizes the repeats and adds more repeats every time a cell replicates its DNA.! ! ! Telomeres also identify ends of chromosomes rather than dsDNA breaks!

Telomerase linked to both cancer and aging.!

Lagging strand cannot be completed !

*!

Must have a base-paired residue with a 3 hydroxyl to be synthesized by the DNA polymerase! ! Primase requires ~ 20 base pairs to generate a 10 base pair primer! ! ! At some point, there is not enough room left on the template strand for the primase!

*!

!"
Figure 6-18 Essential Cell Biology ( Garland Science 2010)

Failure to repair DNA mistakes can have serious consequences !

! Can lead to permanent changes in the DNAmutations. !

Ex. Sickle Cell Anemia !

A DNA mismatch repair system removes replication errors that escape DNA polymerase proofreading!
! DNA mismatch repairthe backup system! !
! Fixes DNA mismatches left behind by replication machine.! ! Pretty effective (>99%), but not perfect! !

Because germline mutations result in an entire organism having mutation, protecting the germ cells from mutations is critical!
! ! ! ! ! Germ cellsthe reproduction cells! = sperm and egg ! (ex. genetic diseases like SCA)! Somatic cellsevery other cell in ! your body (ex. cancer*)!
Due largely to the accumulation of mutations over time. Anything that speeds up this process could be disastrous (ex. Mutation or deletion of DNA repair enzyme).! ! colon cancer in women!

*!

Mismatches must be repaired properly to avoid mutations !

*!

bad !

worse !

good !

! In eukaryotes, still not known how DNA repair machinery tells the difference between the 2 strands. New DNA might be nicked (ss breaks).!

*!
BAD!

Figure 6-21a Essential Cell Biology ( Garland Science 2010)

*!
GOOD!

GOOD!

Figure 6-21c Essential Cell Biology ( Garland Science 2010)

*!
BAD!

WORSE!

Figure 6-21b Essential Cell Biology ( Garland Science 2010)

Mismatches must be repaired properly to avoid mutations !

bad !

worse !

good !

! In eukaryotes, still not known how DNA repair machinery tells the difference between the 2 strands. New DNA might be nicked (ss breaks).!

DNA mismatch repair !

*!

DNA mismatch repair !

*!

Distorts dsDNA; hence can be recognized as different!

Nick identies the newly synthesized strand!

Spontaneous events that compromise DNA integrity!

*!

Figure 6-23 Essential Cell Biology ( Garland Science 2010)

Spontaneous events that compromise DNA integrity!

*!

Spontaneous events that compromise DNA integrity!

*!

If not xed, can lead to mutations !

Thymine Dimers can form as consequence of UV radiation!

*!

Figure 6-24 Essential Cell Biology ( Garland Science 2010)

Damaged DNA can repair itself using its backup copy !

! Can use complementary strand as template.! ! ! Since most DNA damage creates strange looking structures, easy to differentiate the two strands.! ! Proteins (nucleases) involved in Step 1 vary with different types of DNA damage.! ! Base Excision Repair (BER) System!

*!

What happens when both strands of DNA are damaged? !

! Can happen from ionizing radiation, replication fork errors, various chemicals and metabolites, etc.! ! Nonhomologous end-joining (NHEJ) is the most common mechanism to repair dsDNA breaks in somatic cells.! ! Usually OK since most of genome non-coding.! ! Quick and dirty.!

*!

Homologous Recombination !
! Can produce error-free repairs.! ! ! Involves using entirely separate DNA duplex (ex sister chromatids) to x dsDNA break.! ! Also used extensively to produce genetic diversity during meiosis (swapping between maternal and paternal chromosomes). ! ! Requires extensive stretches of sequence similarity (homology). But doesnt have to be absolutely perfect homology.! ! Donor DNA needs to be in close proximity following dsDNA break.! ! Versatile DNA repair mechanism. Highly conserved.!

*!

Homologous Recombination !

*!

Homologous recombination during meiosis !

! During meiosis, chromosomal crossovers lead to the exchange of genetic information.! ! During meiosis recombination preferentially occurs between maternal and paternal chromosomes rather than between newly replicated, identical DNA strands like when HR repairs dsDNA breaks.!
(meiosis-specic proteins) !

Homologous recombination during meiosis !

! Can lead to 2 different types of recombination.!

*!
! If heteroduplex has any mismatches, DNA can undergo mismatch repair.!

more common !

less common !

*!

*!

Gene Conversion !

Crossover !

Discovery of Genetic Transposition!

Jumping Genes! Transposons !

*!

Barbara McClintock! (1902 1992)!

Mobile Genetic Elements (Transposons) !

! jumping genes (molecular parasites ?)! ! Short specialized sequences of DNA that can move throughout a cells genome.! ! Can carry other genes.! ! ! Responsible for much more rapid evolutionary genetic changes.! ! Typically affect only that cell and its descendants.! ! ! Can be major cause of antibiotic resistant bacteria.!

*!

Mobile Genetic Elements (Transposons) !

*!

inverted repeats!

5---GACTGCGCAGTC---3!

Mobile Genetic Elements encode the components they need for movement !

*!

! Unlike HR, dont require sequence homology.! ! Contains ! !(1) Gene for transposase (catalyzes the movement of that element via specialized recombination)! !(2) DNA sequences that are recognized by its transposase.! ! ! Nearly half of human genome! ! is occupied by millions of ! !copies of various mobile ! !genetic elements!!!

Figure 6-33 Essential Cell Biology ( Garland Science 2010)

Human genome contains 2 major families of transposable elements !

1.! DNA-only transposons!
!

*!

2.! Retrotransposons!
! ! ! Uses RNA intermediate! Unique to eukaryotes! Most common type!

! L1 element (LINE-1); 15% human genome! ! Alu sequence; ~1 million copies in our genome; dont encode their own reverse transcriptase! ! Both proliferated in primates relatively recently.!

Alu Sequence Distribution!

*!

Arthrobacter luteus restriction endonuclease! ! ~ 300 bps! ! Formed from the 7SL RNA component ! of the Signal Recognition Particle!

Viruses: the ultimate mobile DNA !

Figure 6-37 Essential Cell Biology ( Garland Science 2010)

Viruses: the ultimate mobile DNA !

! Essentially strings of genes wrapped in a protein coat. Very small.! ! Parasitesthey need to use cells machinery to replicate.! ! Often lethal (ex lytic) to cell.!

Retroviruses are found only in eukaryotic cells.!

Viruses commandeer the host cells machinery !

*!

This lysis can cause an immune response. !

Retroviruses make DNA from an RNA template using reverse transcriptase !

*!

Figure 6-38 Essential Cell Biology ( Garland Science 2010)

Retroviruses make DNA from an RNA template using reverse transcriptase ! e.g., HIV!
Major drug target for AIDS since unique to virus !

*!

Latent phase; virus can hide for a long time!

Lytic phase !

End!

Essential Cell Biology

Third Edition

Chapter 7 From DNA to Protein: How Cells Read the Genome !

Copyright Garland Science 2010

Charles Robert Darwin !

Alfred Russell Wallace !

Francis Harry C. Crick!

James Dewey Watson!

The Central Dogma of Molecular Biology !

! Occurs in all cells from bacteria to humans.! ! ! One of the dening characteristics of living cells.! ! Allows massive amplication of signals from a single gene.!

*!

Caught in the Act!

Actively Transcribing Vertebrate DNA!

Figure 7-8 Essential Cell Biology ( Garland Science 2010)

The efciency of gene expression is quite variable !

! Translation efciency, as well as RNA and protein stability vary greatly among genes!

Structure of RNA !
Differs from DNA in 2 signicant ways:! ! 1. ribonucleotides vs deoxyribnucleotides! ! 2. uracil vs thymine!

RNA is typically single stranded !

! Because RNA is single stranded, intramolecular base pairing can occur, resulting in elaborate secondary structure! ! This gives rise to diverse functionality (e.g., ribozymes, riboswitches, tRNA, rRNA, )!

RNA serves many functions !

Gene expression refers to the biosynthesis of either DNA-encoded protein, or non-coding RNA !

*!

nal product = RNA molecules !

From an evolutionary perspective, RNA may have been the original, self-replicating biopolymer! ! ! Ribozymes may have developed the ability to direct protein synthesis! ! ! DNA is probably a relative newcomer, usurping RNAs role in information storage!

Transcription is the DNA-directed biosynthesis of a single, complementary RNA molecule !

! ! ! ! RNA is much shorter than DNA.! RNA polymerase carries out transcription.! RNA polymerase does not need a primer.! Many RNA polymerases can transcribe a single gene at the same time.!

! Transcription does have similarities to replication.! ! ! As for DNA, RNA is synthesized in the 5 to 3 direction.!

*!

Figure 7-7 Essential Cell Biology ( Garland Science 2010)

Eukaryotic transcription differs from bacterial transcription in a few ways !

1.! Bacteria have only 1 RNA pol. Eukaryotes have 3.!

*!

* !

2.! Eukaryotic RNA polymerases require a bunch of accessory proteins called the general transcription factors (GTFs) to initiate transcription.! 3.! Control mechanisms are more complex in eukaryotes in part because genes are much further apart. This allows more sophisticated gene regulation.! 4.! Eukaryotic transcription has to deal with more compact chromatin structure.!

Bacterial promoters and terminators have specic sequences recognized by RNA polymerase !
! The promoter orients RNA pol and tells it where to start and which way to go.! ! ! All bacterial genes have promoter and terminator sequence similar to those shown below.!

Certain sequences of DNA tell RNA polymerase where to start (promoters) and stop (terminator/stop site) !
! ! In bacteria, the sigma factor, a subunit of the RNA pol, recognizes the promoter. ! ! This is a dynamic process.!

*!

In bacteria !

Bacterial Transcription!

*!

Figure 7-9 Essential Cell Biology ( Garland Science 2010)

Either strand of DNA can act as a template, but the promoter is asymmetrical !

The General Transcription Factors !

! ! ! ! Assemble on the promoter.! Position RNA polymerase.! Open DNA.! Launch the RNA polymerase.!
typically ~25 bp upstream of start site; most promoters have this ! Both opens DNA and phosphorylates and releases RNA pol from initiation complex !

TBP is a subunit of TFIID that distorts DNA, forming landmark !

*!

transcription initiation complex !

! All components are then later recycled to be used again and again!

~ 25 bp upstream from transcription start site!

TBP distorts DNA!

*!

TFIIB provides scaffold!

TFIIH separates strands!

Figure 7-12 (part 1 of 2) Essential Cell Biology ( Garland Science 2010)

TATA Box Binding Protein distorts the double helix!

*!

Figure 7-13 Essential Cell Biology ( Garland Science 2010)

~ 25 bp upstream from transcription start site!

TBP distorts DNA!

TFIIB provides scaffold!

TFIIH separates strands!

Figure 7-12 (part 1 of 2) Essential Cell Biology ( Garland Science 2010)

*!

TFIIF phosphorylates tail ! domain of RNA pol II ! ! ! ! ! ! . launches polymerase!

Figure 7-12 (part 2 of 2) Essential Cell Biology ( Garland Science 2010)

*!
Control of transcription initiation is the most common way organisms regulate control gene expression. !

Eukaryotic RNAs must be processed and exported to cytoplasm!

! In eukaryotes, transcription occurs in nucleus, translation occurs in cytoplasm. The export of RNA occurs via nuclear pore complexes in the nuclear envelope.! ! ! Prior to nuclear export, RNA processing occurs as the RNA molecule is being synthesized.! !

RNA processing !
! Occurs as RNA is being made.! ! Processing machinery is recruited to the phosphorylated tail ! !domain of the eukaryotic RNA polymerase.! ! Different types of processing occurs depending of what type of RNA is being made. ! !

*!

*!

Figure 7-15 Essential Cell Biology ( Garland Science 2010)

Eukaryotic mRNA processing !

! Addition of 5-caps and 3polyadenylation tails (poly-A tails) (also intron splicing) .! ! 5-caps and poly-A tails:!
1.! Increase stability.! 2.! Identies the molecule as mRNA.! 3.! Marks the mRNA as being complete.!

*!

Usually gets trimmed back rst before few hundred A added. !

! !Eukaryotic genes are often interrupted by noncoding sequences (introns). Need to remove/splice these introns out to get nished/meaningful message.! ! Exons-expressed sequences! ! Introns-intervening/nonexpressed sequences!

Introns in eukaryotic mRNA are removed by RNA splicing !

*!

Splicing can happen in prokaryotes, but rarely does.!

Introns are removed by RNA splicing !

! ! ! Occurs during transcription after 5capping. Can occur before during or after addition of poly-A tail.! Involves cutting out introns and stitching exons back together.! Unlike exons, most of intron sequence appears to be unimportant. There are a few short sequences at or near each intron end that act as cues for removal.!

*!

carried our primarily by catalytic RNA molecules (small nuclear RNAs; snRNAs) coupled to a few proteins to form small nuclear ribonucleoprotein particles (snRNPs)forming the core of the Spliceosome.!

*!

This process carried our primarily by catalytic RNA molecules (small nuclear RNAs; snRNAs) coupled to a few proteins to form small nuclear ribonucleoprotein particles (snRNPs)forming the core of the Spliceosome.!

snRNPs bind to specic sequences at both ends of the intron.! ! The 2 hydroxyl of a conserved A attacks 5 splice site forming lariat! ! 3 hydroxyl of rst exon attacks 3 splice site, knitting exons together! ! Lariat is degraded!
Figure 7-20 Essential Cell Biology ( Garland Science 2010)

*!

Introns are removed by RNA splicing !

*!

! Alternative splicing leads to greater protein diversity from single gene.! ! ~60% of human genes undergo alternative splicing. ! ! Could have helped speed evolution of eukaryotes ! !(e.g., domain swapping ).!