BIOCHEMISTRY—MOLECULAR
Chromatin Negatively charged DNA loops twice around Think of beads on a string. structure histone octamer (2 each of the positively charged H2A, H2B, H3, and H4) to form nucleosomebead. H1 ties nucleosomes together in a string (30-nm H1 is the only histone that is not fiber). in the nucleosome core.  In mitosis, DNA condenses to form mitotic chromosomes. Heterochromatin Condensed, transcriptionally inactive. Euchromatin Less condensed, transcriptionally active. Eu=true, “truly transcribed.”
Nucleotides Purines (A, G) have 2 rings. Pyrimidines (C, T, U) PURe As Gold: PURines. have 1 ring. Guanine has a ketone. Thymine has CUT the PY (pie): a methyl. Deamination of cytosine makes uracil. PYrimidines. Uracil found in RNA; thymine in DNA. THYmine has a meTHYl. G-C bond (3 H-bonds) stronger than A-T bond (2 H-bonds). ↑ G-C content → ↑melting temperature.
Amino acids necessary for purine synthesis: Glycine Aspartate Glutamine De novo nucleotide synthesis: Purines are made from IMP precursor. Pyrimidines are made from Nucleotides (base + ribose + phosphate) are linked by orotate precursor, with 3′-5′phosphodiester bond. PRPP added later. Nucleoside = base + ribose. Ribonucleotides are synthesized first and are converted to deoxyribonucleotides by ribonucleotide reductase.
HIGH-YIELD PRINCIPLES BIOCHEMISTRY
Aspartate
Purine (A, G) Pyrimidine (C, T, U)
CO2
Glycine
N
C
N
C
C
C
N
N
C
Glutamine
N10–Formyl- tetrahydrofolate
N10–Formyl- tetrahydrofolate
N
C
N
C
C
C
Carbamoyl phosphate
Aspartate
Nucleosome core histones H2A, H2B, H3, H4
DNA Histone H1
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BIOCHEMISTRY—MOLECULAR (continued)
Transition vs. transversion Transition Substituting purine for purine or pyrimidine for TransItion = Identical type. pyrimidine. Transversion Substituting purine for pyrimidine or vice versa. TransVersion = conVersion between types.
Genetic code features Unambiguous Each codon specifies only 1 amino acid. Degenerate/ More than 1 codon may code for the same Methionine encoded by only redundant amino acid. 1 codon (AUG). Commaless, Read from a fixed starting point as a continuous Some viruses are an exception. nonoverlapping sequence of bases. Universal Genetic code is conserved throughout evolution. Exceptions include mitochondria, archaebacteria, Mycoplasma, and some yeasts.
Mutations in DNA Silent Same aa, often base change in 3rd position of Severity of damage: nonsense codon (tRNA wobble). >missense >silent. Missense Changed aa (conservative––new aa is similar in chemical structure). Nonsense Change resulting in early stop codon. Stop the nonsense! Frame shift Change resulting in misreading of all nucleotides downstream, usually resulting in a truncated protein.
BIOCHEMISTRYHIGH-YIELD PRINCIPLES
83
DNA replication Eukaryotes Eukaryotic genome has multiple origins of Eukaryotic DNA replication is replication. more complicated than the Replication begins at a consensus sequence of prokaryotic process but uses AT-rich base pairs. many enzymes analogous to those below. Prokaryotes Single origin of replication––continuous DNA replication is bidirectional DNA synthesis on leading semiconservative. strand and discontinuous (Okazaki fragments) on lagging strand. Replication fork   Y-shaped region along DNA template where leading and lagging strands are synthesized. Helicase              Unwinds DNA template at replication fork. Single-stranded binding (SSB) protein prevents strands from reannealing. DNA Create a nick in the helix to relieve supercoils. Fluoroquinolones inhibit DNA topoisomerases DNA gyrase is a specific prokaryotic topoisomerase. gyrase. Primase Makes an RNA primer on which DNA polymerase III can initiate replication. DNA polymerase Elongates the chain by adding DNA polymerase III has III deoxynucleotides to the 3′end (leading strand). 5′→ 3′synthesis and Elongates lagging strand until it reaches primer proofreads with 3′→ 5′ of preceding fragment. 3′→5′ exonuclease exonuclease. activity “proofreads” each added nucleotide. DNA polymerase I and III are prokaryotic. DNA polymerase I Degrades RNA primer and fills in the gap DNA polymerase I excises with DNA. RNA primer with 5′→3′ DNA ligase Seals. exonuclease.
HIGH-YIELD PRINCIPLES BIOCHEMISTRY
3'
5'
3'
5'
5'
   3'
Leading strand
Primase
Okazaki fragment DNA ligase
Lagging strand
RNA primerDNA polymerase III
Helicase
Replication fork
Single-strand binding protein
DNA polymerase III
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BIOCHEMISTRY—MOLECULAR (continued)
DNA repair Single strand Nucleotide excision Specific endonucleases release the oligonucleotide- Mutated in xeroderma repair containing damaged bases; DNA polymerase and pigmentosum(dry skin with ligase fill and reseal the gap, respectively. melanoma and other cancers, Base excision repair Specific glycosylases recognize and remove damaged “children of the night”), which bases, AP endonuclease cuts DNA at apyrimidinic prevents repair of site, empty sugar is removed, and the gap is filled thymidine dimers. and resealed. Mismatch repair Unmethylated, newly synthesized string is recognized, Mutated in hereditary mismatched nucleotides are removed, and the gap is nonpolyposis colorectal filled and resealed. cancer(HNPCC). Double strand Nonhomologous Brings together two ends of DNA fragments. end joining No requirement for homology.
DNA/RNA/protein DNA and RNA are both synthesized 5′→3′. mRNA is read 5′to 3′. Protein  synthesis direction Remember that the 5′of the incoming nucleotide  synthesis is N to C. bears the triphosphate (energy source for bond). The 3′hydroxyl of the nascent chain is the target.
Types of RNA Of mRNA, rRNA, and tRNA: Massive, Rampant, Tiny. mRNA is the longest type. rRNA is the most abundant type. tRNA is the smallest type.
Start and stop codons mRNA initiation AUG (or rarely GUG). AUG inAUGurates protein codons synthesis. Eukaryotes Codes for methionine, which may be removed before translation is completed. Prokaryotes Codes for formyl-methionine (f-Met). mRNA stop codons UGA, UAA, UAG. UGA = U Go Away. UAA = U Are Away. UAG = U Are Gone.
Regulation of gene expression Promoter Site where RNA polymerase and multiple other Promoter mutation commonly transcription factors bind to DNA upstream from results in dramatic ↓ in gene locus (AT-rich upstream sequence with amount of gene transcribed. TATA and CAAT boxes). Enhancer Stretch of DNA that alters gene expression by binding transcription factors. May be located close to, far from, or even within (in an intron) the gene whose expression it regulates. Silencer Site where negative regulators (repressors) bind.
BIOCHEMISTRYHIGH-YIELD PRINCIPLES
85
Functional organization of the gene
RNA polymerases Eukaryotes RNA polymerase I makes rRNA. I, II, and III are numbered as RNA polymerase II makes mRNA. their products are used in RNA polymerase III makes tRNA. protein synthesis. No proofreading function, but can initiate chains. RNA polymerase II opens DNA at promoter site. α-amanitin inhibits RNA polymerase II. α-amanitin is found in death Prokaryotes RNA polymerase (multisubunit complex) makes cap mushrooms. all 3 kinds of RNA.
RNA processing Occurs in nucleus. After transcription: Only processed RNA is (eukaryotes) 1. Capping on 5′end (7-methylguanosine) transported out of the 2. Polyadenylation on 3′end (≈ 200 A’s) nucleus. 3. Splicing out of introns AAUAAA = polyadenylation Initial transcript is called heterogeneous nuclear signal. RNA (hnRNA). Poly-A polymerase does not Capped and tailed transcript is called mRNA. require a template.
Splicing of pre-mRNA Pre-mRNA splicing occurs in eukaryotes. 1—Primary transcript combines with snRNPs and other proteins to form spliceosome. 2—Lariat-shaped intermediate is generated. 3—Lariat is released to remove intron precisely and join 2 exons.
Introns vs. Exons contain the actual genetic information INtrons stay IN the nucleus, exons coding for protein. whereas EXons EXit and are Introns are intervening noncoding segments of DNA. EXpressed.
Different exons can be combined by alternative splicing to make unique proteins in different tissues (e.g.,β-thalassemia mutations).
HIGH-YIELD PRINCIPLES BIOCHEMISTRY
DNA
mRNA
Exons
Introns
Transcription and splicing
HO-AAAA
3'
5'
Cap Gpppp
Tail
Coding
GU A AG
–OH UGA AG
exon 1
exon 1exon 2
exon 2
1
2
3
exon 1
exon 1
GU AG exon 2
exon 2
UGA
A
Transcription initiation site Coding regionEnhancer Promoter Exon IntronPromoter TATA AATAAA 5′ 3′
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BIOCHEMISTRY—MOLECULAR (continued)
tRNA Structure 75–90 nucleotides, 2º structure, cloverleaf form, anticodon end is opposite 3′aminoacyl end. All tRNAs, both eukaryotic and prokaryotic, have CCA at 3′end along with a high percentage of chemically modified bases. The amino acid is covalently bound to the 3′end of the tRNA. Charging Aminoacyl-tRNA synthetase (1 per aa, uses ATP) Aminoacyl-tRNA synthetase scrutinizes aa before and after it binds to tRNA. If and binding of charged incorrect, bond is hydrolyzed by synthetase. The tRNA to the codon are aa-tRNA bond has energy for formation of peptide responsible for accuracy of bond. A mischarged tRNA reads usual codon but  amino acid selection. inserts wrong amino acid.
tRNA wobble Accurate base pairing is required only in the first 2 nucleotide positions of an mRNA codon, so codons differing in the 3rd “wobble” position may code for the same tRNA/amino acid.
Protein synthesis
Initiation Activated by GTP hydrolysis, initiation factors (eIFs) Eukaryotes––80S→60S+40S help assemble the 40S ribosomal subunit with the (Even). initiator tRNA and are released when the mRNA Prokaryotes––70S →50S + 30S and the ribosomal subunit assemble with the (odd). complex. ATP––tRNA Activation Elongation 1. Aminoacyl-tRNA binds to A site except for (charging). initiator methionine GTP––tRNA Gripping and 2. Peptidyltransferase catalyzes peptide bond Going places (translocation). formation, transfers growing polypeptide to amino acid in A site A site = incoming Aminoacyl 3. Ribosome advances 3 nucleotides toward 3′end of tRNA. RNA, moving peptidyl RNA to P site (translocation) P site = accommodates growing Termination Completed protein is released from ribosome Peptide. through simple hydrolysis and dissociates. E site = holds Empty tRNA as it Exits.
BIOCHEMISTRYHIGH-YIELD PRINCIPLES
Met
ATP         AMP + PPi
OH 3'
5'
Aminoacyl-tRNA synthetase
Aminoacyl-tRNA synthetase
mRNA Codon
Anticodon (CAU) UAC
AUG
Met
3'
3'
5'
5'
3'5'
Ribosome
40S
E P A
60S
87
Energy requirements of translation tRNA aminoacylation ATP →AMP (2 phosphoanhydride bonds) Loading tRNA onto ribosome GTP →GDP Translocation GTP →GDP Total energy expenditure 4 high-energy phosphoanhydride bonds
Posttranslational modifications Trimming Removal of N- or C-terminal propeptides from zymogens to generate mature proteins. Covalent alterations Phosphorylation, glycosylation, and hydroxylation. Proteasomal Attachment of ubiquitin to defective proteins to tag them for breakdown.