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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
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
Section 18.1: Genetic Information: Replication, Repair, and Recombination
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
Section 18.1: Genetic Information: Replication, Repair, and Recombination
During DNA synthesis, DNA fragments— are joined together by DNA ligase, which catalyzes the formation of the phosphodiester bond between adjoining nucleotides
Section 18.1: Genetic Information: Replication, Repair, and Recombination
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
Section 18.1: Genetic Information: Replication, Repair, and Recombination
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
Section 18.1: Genetic Information: Replication, Repair, and Recombination
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
Section 18.1: Genetic Information: Replication, Repair, and Recombination
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
Section 18.1: Genetic Information: Replication, Repair, and Recombination
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
Section 18.1: Genetic Information: Replication, Repair, and Recombination
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
Section 18.1: Genetic Information: Replication, Repair, and Recombination
Section 18.1: Genetic Information: Replication, Repair, and Recombination
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
Section 18.1: Genetic Information: Replication, Repair, and Recombination
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
Section 18.1: Genetic Information: Replication, Repair, and Recombination
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
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
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
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
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
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
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
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