Ch.24 Genes and Chromosomes
Organization of information in chromosomes
DNA supercoiling
Structure of the chromosome
Molecular Coding of Protein Sequence Information
In transcription, one strand of double-stranded DNA acts as the molecular template for RNA synthesis (DNA -5 messenger RNA).
In translation, the triplets of nucleotides in mRNA bind to complementary triplets in tRNA.
— Three nucleotides code for one 'tcodon."
— The tRNA molecules carry an amino acid associated with the particular triplet.
— Amino acids are then assembled in peptide chains. Protein sequence determines its biological function.
Bacterial Genomes Are Double-Stranded Circles
E.coli: 4,639,675 bp
Bacteria also contain extra-chromosomal, double-stranded circular plasmids.
— 850x longer than the cell!
—usually 0000-10,000 bp, but can be 400,000 bp
— swapped easily between bacteria
— no essential genes, but Often encode genes that degrade antibiotics
— plasmid exchange: one way bacteria acquire antibiotic resistance
Eukaryotic Chromosome
A pair of linked and condensed sister chromatids of a human chromosome.
Eukaryotic chromosomes are in this state after replication at metaphase during mitosis.
Mitochondrial DNA
• It codes for mitochondrial rRNAs tRNAs and some of the mitochondrial specific proteins.
• Most mitochondrial proteins (at least 95%) are encoded by nuclear genes.
types of RNA
DNA Is Packaged with Proteins
• Viral genomic DNA may be associated with capsid proteins.
• Prokaryotic DNA is associated with proteins in the nucleoid.
• Eukaryotic DNA is organized with proteins into a complex called chromatin.
Composition of the Human Genome
Notice that only a small fraction (1.5%) of the total genome encodes for proteins.
The biological significance of noncoding sequences is not entirely clear.
— Some DNA regions directly participate in the regulation of gene expression (promoters, termination signals, etc.).
— Some DNA encodes for small regulatory RNA with poorly understood functions.
— Some DNA may be junk (pieces of unwanted genes, remnants of viral infections).
Eukaryotic Genes Contain Intervening Sequences (Introns)
Exons are expressed sequences (translated into
amino acid sequence).
Introns are regions of genes that are transcribed but
not translated.
Introns are removed after transcription and the exon
mRNA sequences are spliced together.
— Exons account for only 1.5% of human DNA!
— They do not encode polypeptide sequence.
creates ' mature transcripts
Some Bacterial Genomes Also Contain Introns
• Until 1993, scientists thought that introns appeared only in eukaryotes.
• About 25% of sequenced bacterial genomes show presence of introns.
• Introns in bacterial chromosome do not interrupt protein-coding sequences; thev interrupt mainly tRNA sequences.
• Introns in phage genomes within bacteria interrupt protein-coding sequences.
• Many bacterial introns encode catalytic RNA sequences that have the ability to insert and reverse transcribe themselves into the genomic DNA.
Transposons
Transposons Are Sequences That Can Move Within the Genome
• The eukaryotic genome is not completely static.
• Sequences called transposons can move around within the genome of a single cell.
• The ends of transposons contain terminal repeats.
• These repeats hybridize with complementary regions of target DNA during insertion.
• Transposons account for N50% of the human genome.
Eukaryotes Also Contain Highly Repetitive DNA or Simple Sequence Repeats (SSRs)
Short sequences of 10 bp or less
Repeated millions of times
Also known as "satellite" DNA — because when fragmented and centrifuged, the DNA separates into a discrete "satellite" band
Associated with centromeres and telomeres
Centromere Sequences Are Where Proteins Attach During Mitosis
• Region where the chromatids are held together during mitosis — that is, after DNA replication but before cell division
• Essential for equal distribution of chromosome sets to daughter cells
• Have AT-rich repeated sequences of N 130 bp
Telomere Sequences Cap the Ends of Eukaryotic Chromosomes
• May form special loop structures to keep DNA ends from unraveling — contain multiple repeats with general sequence
• (T G ) (A C ) where n = 1500 or more in mammals — added by enzyme telomerase (protein+nucleotides)
Telomeres and Centromeres in a Yeast Chromosome
Telomeres Are Associated with Cellular Aging
• In many tissues, telomeres are shortened after each round of replication.
• Thus, the cellular DNA "ages."
• Normal human cells divide about 52 times before losing the ability to divide again (Hayflick limit).
DNA Supercoiling
DNA in the cell must be organized to allow:
— packing of large DNA molecules within the cells
— access of proteins to read the information in DNA sequence
There are several levels of organization, one of which is the supercoiling of the double-stranded DNA helix.
Closing DNA in a Loop Introduces Supercoiling
Linking Number (Lk) Describes Supercoiling
In circular DNA, changing the helical turns requires breaking a strand transiently.
Linking number in relaxed DNA: Lk= #bp + #bp/turn
Example:
Relaxed circular dsDNA of 2100 bp in the B form (10.5 bp/turn) has [Lk = 2100 bp + 10.5/turn = 200]
Lk is an integer for closed-circular DNA and is (+), reflecting a right-handed helix.
Linking Number Can Be Broken Down into Twist (Tw) and Writhe (Wr)
Lk = Tw+ Wr
Writhe (Wr) is the # coils
— typically a negative value
Twist (Tw) is the # twists or turns of the helix
Topoisomerase
Underwinding Facilitates Additional DNA Structural Changes
Helps to maintain structure of cruciforms at palindromes (next slide)
Cruciforms rarely occur in relaxed DNA.
Facilitates formation of stretches of left-handed Z form
Topoisomers Are DNAs That Differ Only in Linking Number
• Same # bp, same sequence but different degree of supercoiling
• Conversion between topoisomers requires a DNA strand break.
• Note that negatively supercoiled DNA (more compact) travels faster in an agarose gel electrophoresis experiment than relaxed or nicked DNA do.
Topoisomers in Electrophoresis
Topoisomerases Are Enzymes That Change Lk
These enzymes are required for DNA unwinding and rewinding during transcription and replication.
Two major types:
Type I — make a transient cut in one DNA strand changes Lk by 1
Type Il — make a transient cut in both DNA strands change Lk in steps of 2
The Topoisomerases I—IV of E. Coli
Topo I and Ill are Type I.
Topo Il is called DNA gvrase.
— remove negative supercoils to relax DNA
— increase Lk
— use single-stranded breaks
— introduces negative supercoils
— decreases Lk
— uses ATP and double-stranded breaks
Eukaryotic Topoisomerases Include
Topo l, lla, llß, IV
Topo I and Ill are Type I (as in E. coli).
Type Il topoisomerases include two subfamilies
—Type IIA and Type 11B.
— can relax both positive and negative supercoils
Topoisomerases Are Targets for Antibiotics
Coumarins (novobiocin, coumermycin Al)
Quinolones (nalidixic acid; ciproflaoxadin, Cipro)
— inhibit bacterial Type Il topoisomerases from binding ATP
— inhibit the last step, which is resealing the DNA strand breaks
— wide-spectrum and mostly selective for bacterial enzymes
Topoisomerase Inhibitors Used as Chemotherapy Agents
Eukaryotic Type I topoisomerase inhibitors
Eukaryotic Type Il topoisomerase inhibitors
Targets cancer because most rapidly growing cells (tumors, others) express topoisomerases
Supercoiled DNA Forms Plectonemic or Toroid/Solenoid Structures
(or a Combination)
Plectonemic
Toroid/solenoid
— seen in plasmids
— involves a right-hand superhelix with terminal loops
— used in chromatin
— involves tight left-hand turns
— can resemble a garden hose on a reel
— provides more compaction
Changes in Chromosome Structure During the Cell Cycle
DNA Packing into Chromatin
• Chromatin consists of fibers of protein and DNA and a small amount of RNA.
• DNA associates tightly with proteins called histones.
(post-translational modification)
• DNA and protein are packed into discrete units called nucleosomes.