Chapter 18 Section 18.1: Section 18.1: Genetic Information: Replication, Repair, and Recombination Section 18.2: Section 18.2: Transcription Section 18.3:

Chapter 18

Section 18.1: Genetic Information: Replication, Repair, and Recombination

Section 18.2: Transcription Section 18.3: Gene ExpressionBiochemistry in the LabBiochemistry in Perspective

Genetic Information

Overview

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Information of Life All information-based systems involve conservation

and transfer of that information DNA is the relatively stable structure that

maximizes information storage and duplication (conservation)

RNA more reactive than DNA (transfers information) Plays numerous roles in protein synthesis and

gene expression Numerous proteins required to maintain and

decode DNA Sequence-specific binding occurs in major and

minor grooves


Chapter 18: Overview

DNA-binding proteins share similar structural features

Most possess a twofold axis of symmetry and can be separated into families:

1. Helix-turn-helix2. Helix-loop-helix3. Leucine zipper4. Zinc finger

Figure 18.1 DNA-Protein Interactions



Ex. Leucine zipper transcription factors form dimers as their leucine-containing a-helices associate via van der Waals forces

Figure 18.1 DNA-Protein Interactions



All living organisms synthesize new DNA rapidly and accurately

They also produce mechanisms to protect and repair DNA, although variations in information can occur

Variation may also be important for adaptability to environmentsVariation is caused by genetic recombination and

mutation



DNA ReplicationDNA replication occurs before

cell division the mechanism is similar in all

living organisms After the two strands have

separated, each serves as a template for synthesis of a complementary strand (semiconservative replication)

Figure 18.2 Semiconservative DNA Replication



DNA Synthesis in Prokaryotes—DNA replication in E. coli consists of several basic steps:DNA unwinding Primer synthesisDNA polynucleotide synthesis



DNA unwinding DNA duplex is unwound by the action of ATP-

dependent helicases (bind to, separate strands)



Primer synthesis: short disconnected RNA segments (primers) are formed.

Catalyzed by primase (an RNA polymerase) DNA polymerases use the primers as starting

point for complementary DNA synthesis (5′ 3′)



DNA polymerase III (pol III) is the major DNA polymerase in prokaryotes

Pol III composed of at least 10 subunits The core polymerase is formed of three subunits: a, e, and The b-protein (sliding clamp) is two subunits and forms a donut-

shaped ring around the template DNA



The g complex is composed of g, d, d, c, and Acts as the clamp-loader, loading b2-clamp

dimer b2-Clamp prevents dissociation of polymerase from

the DNA template The g-complex is ejected in an ATP-dependent

process and replication can proceed



The DNA replicating machine (replisome) consists of two pol III holoenzymes, the primosome (complex of primase and other proteins), and DNA unwinding proteins

There are four other DNA polymerases: DNA polymerase I: Helps remove RNA primer,

replace it with DNA DNA polymerase II, IV, and V: Involved in DNA

repair All three are part of the global SOS response

that prevent cell death due to high levels of DNA damage



During DNA synthesis, DNA fragments— are joined together by DNA ligase, which catalyzes the formation of the phosphodiester bond between adjoining nucleotides



DNA topoisomerases prevent tangling of DNA strands and relieve torque in the DNA, so the replication process is not slowed

Type I topoisomerases produce transient single-strand breaks

Type II topoisomerases produce transient double-strand breaks DNA gyrase—a type II topoisomerase in prokaryotes

helps separate the replication products and create the negative (-) supercoils required for genome packaging



In E. coli when the ATP/ADP ratio is high and there is enough DnaA, replication can begin at the chromosome initiation site (oriC)

Replication proceeds in both directions with each replication fork having helicases and a replisome

E. coli only has one origin of replication, making it a single replication unit (replicon) organism



DNA synthesis only occurs in the 5′3′ direction, so one strand is continuously synthesized (leading strand) while the other is not (lagging strand) The lagging strand is synthesized in short 5′3′

segments called Okazaki fragments (1,000–2,000 nucleotides)

Figure 18.8 DNA Replication at a Replication Fork



DNA replication begins when DnaA proteins bind five to eight 9-bp sites (DNA boxes) within the oriC Oligomerization of DnaA results in a nucleosome-like

structure requiring ATP and histone-like protein (HU) Causes three 13-bp repeats near the DnaA-DNA complex

to open



DnaB complexed with DnaC enters the open oriC region; once DnaB is loaded, DnaC is released The replication fork moves forward as DnaB

unwinds the helix Topoisomerases relieve torque ahead of the

replisome Single strands are kept apart by numerous copies

of single-stranded DNA-binding protein (SSB)

Figure 18.9 Replication Fork Formation



For pol III to initiate DNA synthesis an RNA primer must be present On the leading strand, only a single primer is

required On the lagging strand, a primer is required for each

Okazaki fragment

Figure 18.10 E. coli DNA Replication Model



Pol III synthesizes at the 3′ end of the primerRNA primers are removed by pol I, which then

synthesizes complementary DNADNA ligase then joins Okazaki fragments

Figure 18.10 E. coli DNA Replication Model



DNA replication is fast and accurate: In E. coli, 1,000 base pairs are replicated per second per replication fork, with an error rate between 1/109 - 1/1010 base pairs This is due to the precise nature of the copying

process (complementary), proofreading mechanism of DNA pol I and III, and postreplication repair mechanisms



Replication ends when the replication forks meet at the other side of the circular chromosome at the termination site (ter region) The DNA-binding protein tus binds to the ter

causing replication arrest

Figure 18.11 Role of Tus in DNA Replication Termination in E. coli



DNA Polymerase There are 15 eukaryotic DNA polymerases Three (a, d, and e) are

involved in nuclear DNA replication

Pol γ replicates and repairs mitochondrial DNA

Polymerases b (beta), z(zeta) and h (eta) function in nuclear DNA repair



DNA Synthesis in Eukaryotes has a great deal in common with prokaryotes; they also have significant differences

Timing of replication—eukaryotic replication is limited to the S phase of the cell division cycle

Replication rate is slower in eukaryotes (50 bp per second per replication fork) due to complex chromatin structureFigure 18.12 The

Eukaryotic Cell Cycle



Replicons—eukaryotes have multiple replicons (about every 40 kb) to compress the replication of their large genomes into short periods Humans have 30,000 origins

of replication Okazaki fragments are from

100 to 200 nucleotides long

Figure 18.13 Multiple-Replicon Model of Eukaryotic Chromosomal DNA Replication



The Eukaryotic Replication ProcessReplication begins with the

assembly of the preinitiation replication complex (preRC)

Occurs when cyclin-dependent kinase (Cdk) and cell division cycle (Cde) protein levels are low, limiting DNA replication to once per cell cycle

preRC assembly begins when the origin replication complex (ORC) binds to the origin

Figure 18.14 Formation of a Preinitiation Replication Complex



Cdc6 and Cdt1 proteins bind ORC and recruit the MCM (mini-chromosome maintenance) complex (DNA helicase)

PreRC converted to active initiation complex by addition of pol a/primase, pol e, and accessory proteins

Cell cycle regulating kinases then phosphorylate and activate preRC components


McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press; http://www.nature.com/nrg/journal/v8/n8/fig_tab/nrg2143_F1.html

When the initiation complex is active, newly phosphorylated MCM separates the DNA strands Each strand is then stabilized

by replication protein A (RPA)

Pol a/Primase extends each primer by a short segment of DNA, then polymerase d and e continue the process

Replication factor C (RFC), a clamp loader, controls the attachment of polymerase d


McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

When the replication machinery reaches the 3′ end of the lagging strand, there is insufficient space for a new RNA primer This leaves the end of the chromosome without

its complementary base pairs Chromosomes with 3′-ssDNA (single strand DNA)

overhangs are very susceptible to nuclease digestion

Eukaryotes compensate for this with telomerase, a ribonucleoprotein with reverse transcriptase ability



Telomerase has an RNA base sequence used to synthesize a single-stranded DNA to extend the 3′ strand of the telomere Afterward the normal

replication machinery synthesizes a primer and Okazaki fragment

Figure 18.17 Telomerase-Catalyzed Extension of a Chromosome



During normal human aging, the telomeres of somatic cells shorten over time Once telomeres are

reduced to a critical length, chromosome replication cannot occur

Telomere shortening causes cell death

90% of all cancers have hyperactive telomerase

Figure 18.17 Telomerase-Catalyzed Extension of a Chromosome



DNA RepairCells continually monitor for DNA damage, and

possess multiple repair mechanismsMutations are caused by metabolic activities or

environmental exposures on DNA The natural rate of mutation is about 1.0

mutation per 100,000 genes per generationVideo: DNA Repair



https://www.youtube.com/watch?v=sr-ajrgvg5s

Types of DNA repair

Direct Repairs Some DNA damage can be

repaired without the removal of nucleotides Breaks in phosphodiester

linkages can be repaired by DNA ligase

In photoreactivation repair, pyrimidine dimers are restored to original monomeric structure using a photoreactivating enzyme and visible light

Figure 18.18 Photoreactivation Repair of Thymine Dimers



Single Strand Repairs use the complementary, undamaged strand as a template Base excision repair: A DNA glycosylase

cleaves the N-glycosidic linkage between the damaged base and the deoxyribose protein

Individual nucleotides with damaged bases can be removed

Figure 18.19 Base Excision Repair




Mismatch repair is a single-strand repair mechanism that corrects helix distorting base mispairings resulting from proofreading errors or replication slippage Mechanism distinguishes between old and newly

synthesized strands, which are hemimethylated for a brief period of time



Double-strand breaks (DSBs) are especially dangerous for cells because they can result in a lethal breakdown of chromosomes Caused by radiation, Reactive Oxygen species

(ROS), DNA damaging agents, or as result of replication errors

DSBs are repaired by two mechanisms: non-homologous end joining (NHEJ) homologous recombination



DNA RecombinationRecombination: Rearrangement of DNA

sequences by exchanging segments from different molecules

Genetic recombination is a principle source of the variations that make evolution possible

Two types of recombination: General recombination occurs between

homologous DNA molecules (most common during meiosis)

Site-specific recombination—the exchange of sequences only requires short regions of DNA homology (e.g., transposition)

Mechanism of Recombination



https://www.youtube.com/watch?v=8rXizmLjegI

Transcription is a complex process involving a variety of enzymes and associated proteins

https://www.youtube.com/watch?v=SMtWvDbfHLoRNA polymerase is the enzyme that catalyzes the

addition of ribonucleotides in a 5′3′ direction The template strand (-) of DNA is antiparallel

to the new RNA strand The noncoding strand (+) has the same base

sequence as the RNA, except the transcript substitutes uracil for thymine

Figure 18.31 DNA Coding Strand

Section 18.2: Transcription


https://www.youtube.com/watch?v=SMtWvDbfHLo

https://www.youtube.com/watch?v=SMtWvDbfHLo

Transcription consists of three stages: initiation, elongation, and termination Initiation: RNA polymerase binds to the

promoter (regulatory sequence upstream of a gene)

Figure 18.31 Transcription Initiation in E. coli



Two short consensus sequences at -10 (Pribnow box) and -35 are similar among many bacterial species

RNA Polymerase (RNAP) holoenzyme slides down the DNA until it reaches a promoter sequence

Once the RNAP holoenzyme has bound the promoter region, s and b′ break the hydrogen bonds to unwind a short segment of DNA at the Pribnow box

Figure 18.32 Typical E. coli Transcription Unit



The enzyme-promoter complex is now considered “open” and transcription can begin

RNAP catalyzes the addition of the first nucleoside triphosphate and then continues

Once RNAP has synthesized an RNA chain of 10 nucleotides, it has cleared the promoter and s factor is released




Once s factor is released, RNAPs affinity for the promoter site decreases and the elongation phase begins

DNA unwinds ahead of the transcription bubble The transcription bubble is unwound DNA of 12–

14 bp, containing RNA-DNA hybrid


Figure 18.31 Transcription Initiation in E. coli


There are approximately 30 bp in the RNAP at any one time

The active site of the enzyme lies between the b and b′ subunits The nontemplate strand loops away from the

active site into its own channel When both strands emerge, they re-form a

double helix




Growing RNA chain exits through a channel formed by the b and b′ subunits

Unwinding action by RNA polymerase causes positive supercoils ahead of the transcription bubble and negative supercoils behind the bubble Topoisomerases relieve the supercoils

Transcription continues until a termination signal is reached


Figure 18.31 Transcription in E. coli


Two types of transcription termination in bacteria: intrinsic termination and rho-dependent termination

In intrinsic termination, RNA synthesis is terminated by the transcription of an inverted repeat sequence The inverted repeat forms a stable hairpin that

causes the RNA polymerase to slow or stop RNA transcript is released due to weak base-pair

interactions

Figure 18.34 Intrinsic Termination



In rho-dependent termination, RNA synthesis is terminated with the aid of the ATP-dependent helicase rho factor Rho binds to a specific

recognition sequence on the nascent RNA chain, upstream from the termination site

Unwinds the RNA-DNA helix to release the transcript

Figure 18.35 Rho-Dependent Termination



mRNA translation begins as soon as the ribosome binding site is exposed, but rRNA and tRNA are produced from larger transcripts by posttranscriptional processing via RNases

Figure 18.36 Ribosomal RNA Processing in E. coli



Transcription in Eukaryotes Similar to prokaryotic transcription in several

aspects Polymerases are similar in structure and function Initiation factors are distantly related, but perform

similar functionsRegulatory mechanisms differ significantly in both

organisms https://www.youtube.com/watch?v=Embo24dnKQo



https://www.youtube.com/watch?v=Embo24dnKQo

https://www.youtube.com/watch?v=Embo24dnKQo

Chromatin is usually at least partially condensed

For transcription to occur, DNA most be sufficiently accessible for RNA polymerase

Histone tails of nucleosomes are modified by histone acetyl transferases (HATs) to allow access

Histone-DNA contacts are weakened by chromatin remodeling complexes, SWI,SNF, and NURF

Figure 18.37 Chromatin Remodeling



Eukaryotic transcription involves 3 RNA Polymerases RNA polymerase I (RNAPI) transcribes larger

rRNA (28S, 18S, and 5.8S) in the nucleolus RNA polymerase II (RNAPII) produces the

precursors of mRNA, miRNAs and most (small nuclear) snRNA

RNA polymerase III (RNAPIII) is responsible for transcribing the precursors for tRNA, 5S rRNA, U6 snRNA, and the (small nucleolar) snoRNAs

Eukaryotic RNA polymerase needs various transcription factors bound to the promoter to initiate transcription



Eukaryotic promoters- Promoter sequences in eukaryotic DNA are larger, more complex, and more variable than in prokaryotes Each consists of a core promoter which can be

focused or dispersed Focused promoters contain the transcription start

site (TSS) and core promoter elements (CPE) The most studied CPE is the TATA box (25–30 bp

upstream) promoter sequence TATA-binding protein (TBP) a subunit of the

transcription factor TFIID binds the TATA box and is the first step of RNA polymerase assembly



Proximal promoter elements are transcription factor binding sites within 250 bp of the Transcription Start Site

The frequency of transcription initiation is often affected by upstream sites such as the CAAT box and GC box Can also be affected by enhancers that may

be thousands of base pairs upstream



The eukaryotic enzyme RNA polymerase II (RNAP II) catalyzes the transcription of DNA.

The core enzyme contains 12 subunits in humans

RBP1, the largest subunit, forms part of the enzyme’s active site and binds DNA



The mediator is a protein complex is required for the transcription of most RNAP II genes Has three domains: head,

middle, and tail The mediator acts as an adaptor

between RNAP II and transcription factors, which are bound at positive and negative regulatory gene sequences

This sophisticated form of regulation is largely responsible for the intricate gene expression mechanisms

Figure 18.41 The Yeast Mediator-RNA Polymerase II Holoenzyme Complex



Eukaryotic transcription occurs in several phases, Preinitiation complex (PIC) assembly, initiation, elongation and termination PIC assembly begins with binding

of TBP subunit of TFIID to the TATA box.

The transcriptionally active PIC requires other general transcription factors (GTFs) and a mediator

TFIIH acts as an ATP-dependent helicase

Promoter clearance occurs after 23 nt of mRNA are transcribed

Figure 18.44 Preinitiation Complex Formation at a TATA Box



Elongation begins when promoter clearance has been achieved and RNAP II has dissociated from the mediator

Transcription continues past the functional end of the nascent transcript until the poly(A) sequence is reached (5′-AAUAAA-3′)

Several proteins now linked to RNAP II cause termination by cleaving the transcript 10-30 nt downstream of the poly(A) sequence

Poly(A) polymerase then adds a poly(A) tail (100–200 adenylate residues) to the end of the transcript



RNA Splicing Introns are cut out of the primary RNA transcript

and exons are linked together to form a functional product

The number of introns and exons is highly variable among different genes and species

RNA splicing takes place in a 4.8-megadalton RNA-protein complex called the spliceosome

Splicing occurs at certain conserved sequences RNA Splicing



https://www.dnalc.org/resources/3d/rna-splicing.html

In eukaryotic nuclear pre-mRNA transcripts, there are two intron types: GU-AG and AU-AC

In GU-AG introns, 5′-GU-3′ and 5′-AG-3′ are the first and last dinucleotides of the intron, respectively

The splice event occurs in two reactions:1. A 2′-OH of an adenosine nucleotide within the intron attacks a phosphate in the 5′ splice site, forming a lariat

Figure 18.46 RNA Splicing



2. The lariat is cleaved and the two exons joined when the 3′-OH of the upstream exon attacks a phosphate adjacent to the lariat 5′ splice site is the

donor site and the 3′ splice site is the acceptor site

Four active spliceosomes form with each pre-mRNA to form a supraspliceosome







The precise and timely regulation of gene expression is required for handling changing environments, cell differentiation, and intercellular cooperation

Gene expression is regulated at the following levels: genomic control, transcriptional control, RNA processing, RNA editing, RNA transport, and translational controlGene Expression

Section 18.3: Gene Expression

https://www.youtube.com/watch?v=OEWOZS_JTgk

RNA processing—Among the most important types of RNA processing is alternative splicing The joining of different

combinations of exons to form cell-specific proteins

Figure 18.52 RNA Processing



Tropomyosin is a protein found in a wide variety of tissues (skeletal, smooth, and cardiac muscle, fibroblasts, and brain)

The vertebrate tropomyosin gene consists of 13 to 15 exons with five of the exons common in all protein isoforms The remaining exons are alternatively used in

different tropomyosin mRNAs

Figure 18.53 Alternate Splicing of the Tropomyosin Gene



Documents

Chapter 18 Section 18.1: Section 18.1: Genetic Information: Replication, Repair, and Recombination Section 18.2: Section 18.2: Transcription Section 18.3: