Expression DNA
Gene - Transcription
Genes are expressed as encoded proteins in a 2 step process: transcription + translation Central dogma of biology:
DNA RNA protein
Transcription: copy DNA strand making RNA (in nucleus)
Uses ~50 different transcription factors (proteins) and RNA polymerase Locally opens DNA helix, assembles RNA strand following base pairing and re-closes DNA Many different types of RNA are synthesized, including
Messenger (m- later translated into protein); ribosomal (r- builds ribosomes used to synthesize proteins)(4types) transfer (t- carries amino acids to growing protein) (32 types)
Three types of RNA polymerase one for each of above RNA types Most genes are split into segments (exons average 140 nucleotides code for amino acids in protein and introns, much longer up to 500,000 nucleotides code for a.a. not in protein Introns must be cut and spliced out of mRNA
Translation Summary
Translation involves the reading of mRNA nucleotide triplets (codons) to form tRNAs, specific for each amino acid, which then assemble these to form a polypeptide chain (the protein) - (this occurs in the cytoplasm) The specific steps of translation are
Initiation: mRNA is aligned on the ribosome and is read downstream (5' to 3') till the start codon AUG is found Elongation: using the energy in GTP, coded tRNA-amino acid complexes are brought in and the amino acid is covalently attached via peptide bonds to the previous amino acid and the ribosome moves codon by codon along the mRNA repeating this process Termination: when the ribosome reaches the stop codon (UAA< UAG or UGA), a protein release factor causes the polypeptide to be released from the ribosome and the ribosome splits into its subunits for later reassembly and use Animation at http://www.johnkyrk.com/DNAtranslation.html
Post-translational Protein Modification
As proteins are being synthesized in the cytoplasm, they are often (~80%) protected by chaperones to keep them from aggregating so they can fold properly or by chaperonins (~20%), hollow cylinders lined with hydrophobic amino acids into which the newly synthesized protein fits both use ATP energy After some quality control checks, newly synthesized proteins can be altered by:
Co-valent attachment of sugar residues Or phosphate groups Or sulfate groups
Nucleic Acids
DNA and RNA make up 5 15% of the dry weight of cells 4 different sub-units in DNA or RNA
Each nucleotide = base + sugar + phosphoric acid sugar = D-ribose in RNA or 2-deoxy Dribose in DNA base = 2 purines, A + G (with 2-rings) and 3 pyrimidines (C + T or U- in RNA) (with 1-ring) Base + sugar = nucleoside + phosphate group = nucleotide
Nucleic Acid Structure
DNA Primary Structure
Sequence of bases
Secondary Structure
Watson-Crick helix is the standard configuration of DNA Base-base interactions stabilize the DNA Bases are planar pyrimidines are exactly planar, purines are nearly so Principle of Base Pairing: purine pyrimidine A T (2 H bonds) G C (3 H bonds) Genetic info is all in either strand So, A+G = T+C = total bases in either strand and the ratio (A+T)/(G+C) is characteristic of a species it does not depend on cell type or age
Watson-Crick (or B) DNA helix
Base repeat H bonds Pitch = axial repeat
Genetic Code
Codons = triplet of bases 43 = 64 (degenerate)
Other DNA Helices
Left-handed Z
Forms of DNA
Sizes of Nucleic Acids
Shortest RNAs 75 80 residue t-RNAs Largest RNAs 200,000 res. Smallest DNA few 1000 res. Largest DNA 108 res.
DNA sequencing
Human Genome Project
Chromosomes (50 250 million bases) broken into short pieces Fragments (500 bases) separated by gel electrophoresis
In the much-automated Sanger sequencing method (based on Maxim & Gilbert '77), the single-stranded DNA to be sequenced is "primed" for replication a with short complementary strand at one end. This preparation is then divided into four batches, and each is treated with a different replication-halting nucleotide (depicted here with a diamond shape), together with the four "usual" nucleotides. Each replication reaction then proceeds until a reactionterminating nucleotide is incorporated into the growing strand, whereupon replication stops. Thus, the "C" reaction produces new strands that terminate at positions corresponding to the G's in the strand being sequenced. (Note that when long strands are being sequenced the concentration of the reaction-terminating nucleotide must be carefully chosen, so that a "normal" C is usually paired with a G; otherwise, replication would typically stop with the first or second G.) Gel electrophoresis -- one lane per reaction mixture -- is then used to separate the replication products, from which the sequence of the original single strand can be inferred.
Human Genome
With 3.1647 x 109 base pairs, human DNA could code for 107 proteins (ave M ~ 300 aa) But, lots of nonesense coding (50%)+ stop codes average gene = 3000 bp, largest = dystrophin = 2.4 x 106 bp Number of genes (coded proteins) = 30-35,000 (<2% of DNA codes for proteins) Only 3x more proteins than in the fly 99.9% of genes are identical for all people Over 50% of genes have unknown function
DNA structure in chromosomes
Superhelical (Circular) DNA or A Lesson in Topology
Twist (Tw) of DNA refers to the number of turns of the double helix around its axis. So, since normally B-DNA has 10.5 bp/turn, linear B-DNA with N bp has a Tw = N/10.5 If we twist the DNA we can decrease or increase the Tw, and unwind or overwind the DNA to accommodate the twist, the free ends can simply rotate about one another If the DNA is constrained to form a closed circle then the structure can become more complex.
Writhe and Linking Number
With circular DNA, a change in Tw will not be compensated by rotation of free ends and the (closed circular) ccDNA becomes strained rubber band analogy leads to writhing or supercoiling of the circle Wr refers to the coiling of the ds DNA axis Supercoils can be characterized by the superhelical density = Wr/Tw = # supercoils per turn of DNA Tw and W...