DNA Structure

DNA (deoxyribonucleic acid): a self-replicating molecule that is present in nearly all living organisms. It is the carrier of genetic information

• DNA is composed of nucleotides, DNA nucleotides have three distinct parts

  1. Phosphate

  2. Deoxyribose sugar

  3. Nitrogenous bases

     • DNA bases exist in four variants: Adenine (A), Cytosine (C), Guanine (G), Thymine (T)

• Chargaff’s rules

  • The base composition of DNA varies between species

  • In any species the percentage of A and T bases are equal and the percentages of G and C bases are equal

• DNA base pairing: adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C)

• The double-stranded DNA molecule runs antiparallel (their subunits run in opposite directions)

DNA Replication

• Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand

• Origins of replication - location where the two DNA strands are separated, opening up a replication “bubble” is where replication begins

• Replication fork - Y-shaped region where the parental strands of DNA are being unwound

• Topoisomerase - relieves the strain caused by tight twisting ahead of the replication fork by breaking, wiveling, and rejoining DNA strands

• Sequence of synthesizing a new DNA strand:

  1. DNA helicase unzips the double stranded DNA molecule

  2. Single-stranded binding proteins stabilize the individual strands

  3. The enzyme primase assembles RNA primers (5-10 nucleotides) provide a location for polymerase to begin assembling bases

  4. DNA polymerase (pol III) attaches to the primers and assembles a complementary strand of nucleotides to the template strand in a 5’ to 3’ direction

  5. DNA ligase binds DNA strands together

• DNA polymerases add nucleotides only to the free 3’ end of a growing strand; therefore, a new DNA strand can elongate only in the 5’ to 3’ direction

• Along one template strand of DNA< the DNA polymerase synthesizes a leading strand (5’ → 3’) continuously, moving toward the replication fork

• To elongate the other new strand, the lagging strand(3’ → 5´), DNA polymerase must work in the direction away from the replication fork

• The lagging strand is synthesized as a series of segments called Okazaki fragment

• The remaining gaps are joined together by DNA ligase

Protein Synthesis

• RNA (ribonucleic Acid) is chemically similar to DNA, but RNA has a ribose sugar instead of deoxyribose and the base uracil (U) rather than thymine (T)

  • RNA is usually single-stranded

• Transcription is the synthesis of RNA using information in DNA

• Transcription produces messenger RNA (mRNA)

• Translation is the synthesis of a polypeptide, using information in the mRNA

• Ribosomes are the sites of translations

• During transcription, one of the two DNA strands, called the template strand, provides a template for ordering the sequence of complementary nucleotides in an RNA transcript

• During translation, the mRNA base triplets, called codons, are read in the 5’ to 3’ direction.

• RNA codons are a triplet code, a series of nonoverlapping, three-nucleotide segments

  • eg. ACG

Chemistry of Transcription

The three stages of transcription

1. Initiation (starting)

   • Transcription factors mediate the binding of RNA polymerase and the initiation of transcription

   • The TATA box is a sequence of DNA that serves a promoter, which signals a starting point for the RNA polymerase to bind and begin sequencing RNA nucleotides

   • The stretch of DNA that is transcribed is called a transcription unit

2. Elongation (building)

   • Elongation is the addition of RNA nucleotides to an mRNA molecule

   • As RNA polymerase moves along the DNA it untwists the double helix, 10 to 20 bases at a time added to the 3’ end (5’ → 3’)

3. Termination (ending)

   • RNA polymerase II transcribes the polyadenylation signal sequence; the RNA transcript is released 10-35 nucleotides past this polyadenylation sequence. At this stage the sequence is pre-mRNA

• RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and joins together the RNA nucleotides in the 5’ to 3’ direction

• Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before the genetic messages are dispatched to the cytoplasm

• During RNA processing, both ends of the primary transcript are altered

• Each end of a pre-mRNA molecule is modified in a particular way

  • The 5’ end receives a modified G nucleotide 5’ cap

  • The 3’ end gets a poly-A tail

• Introns - the noncoding regions of mRNA also called intervening sequences

• Exons - the other regions of mRNA are usually translated into amino acid sequences

• RNA splicing - removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence

Introns interrupt and Exons get expressed

• mRNA: contains codons from DNA for protein making

• tRNA: carry amino acids on anticodons

• rRNA: RNA in ribosomes

The three stages of translation:

1. Initiation

   • Initiation begins when a small ribosomal subunit binds with mRNA and a special initiator tRNA, translation is initiated with the start codon (AUG)

2. Elongation

   • During elongation, amino acids are added one by one to the previous amino acid at the C-terminus of the growing chain

   • Translation proceeds along the mRNA in a 5’ to 3’ direction

3. Termination

   • Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome

   • The A site accepts a protein called a release factor

   • The release factor causes the addition of a water molecule instead of an amino acid

   • This reaction releases the polypeptide, and the translation assembly then comes apart

All three stages require protein “factors” that aid in the translation process

• A cell translates an mRNA message into protein with the help of transfer RNA (tRNA)

  • the tRNA contains an amino acid at one end and at the other end has a nucleotide triplet, an anticodon, that can base-pair with the complementary codon on mRNA

• Ribosomes are made of large and small ribosomal units which are made of proteins and ribosomal RNAs (rRNAs)

• Ribozymes are RNA molecules that function as enzymes

• A ribosome has three binding sites for tRNA

  • the A site holds the tRNA that carries the next amino acid to be added to the chain

  • the P site holds the tRNA that carries the growing polypeptide chain

  • the E site is the exit site, where discharged tRNAs leave the ribosome

• Free ribosomes (floating in cytoplasm) mostly synthesize proteins that function in the cytosol (stay in the cell)

• Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell

• Polypeptides destined for the ER or for secretion are marked by a signal peptide

• A signal-recognition particle (SRP) binds to the signal peptide

Regulation of Gene Expression

• Gene expression: the process by which instructions in DNA are transcribed into RNA, and then translated into a functional protein

• Regulatory sequences: stretches of DNA that can be used to promote or inhibit protein synthesis

• Regulatory proteins: are active in assisting the promotion or inhibition of protein synthesis

• Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome

• Transcription factors: proteins that promote or inhibit gene expression

  • Cell differentiation is possible because of the presence of various transcription factors working in concert — (Sequential gene expression)

• The regulatory “switch” is a segment of DNA called an operator, usually positioned within the promoter

• An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control

• Repressible operon: an operon that is usually on; binding of a repressor to the operator shuts off transcription

• Inducible operon: an operon that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription

• The operon can be switched off by a protein repressor this prevents gene transcription by binding to the operator and blocking RNA polymerase

• The repressor is the product of a separate regulatory gene

• A corepressor is a molecule that cooperates with a repressor protein to switch an operon off

• A molecule called an inducer inactivates the repressor to turn the lac operon on

Epigenetics

• Chromatin: a complex of DNa and protein, is found in the nucleus of eukaryotic cells

• Proteins called histones are responsible for the first level of DNA packing in chromatin

• A nucleosome consists of DNa wound twice around a protein core of eight histones, two of each of the main histone types

• Epigenetics: the study of heritable changes in gene expression (active versus inactive genes) that do not involve changes to the underlying DNA sequence **a change in phenotype without a change in genotype

• Histone acetylation: acetyl groups are attached to histone tails which generally loosen chromatin structure, promoting the initiation of transcription

• DNA methylation is the addition of methyl groups to certain bases in DNA. Individual genes are usually more heavily methylated in cells where they are not expressed

• After replication, enzymes methylate the correct daughter strand so that the methylation pattern is inherited.

Gene Mutations

• Mutations are changes in the genetic material of a cell or virus

• A nucleotide-pair substitution (point mutations) replaces one nucleotide and its partner with another pair of nucleotides

• Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code

• Missense mutations still code for an amino acid, but not the correct amino acid

• Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein

• Insertions and deletions are additions of losses of nucleotide pairs in a gene

• Insertion or deletion of nucleotides may alter the reading frame of the genetic message, producing a frameshift mutation

Biotechnology techniques and Uses

• Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites

• A restriction enzyme usually makes many cuts, yielding restriction fragments

• The most useful restriction enzymes cleave the DNA in a staggered manner to produce sticky ends

• Sticky ends can bond with complementary sticky ends of other fragments

• To see the fragments produced by cutting DNA molecules with restriction enzymes, researchers use gel electrophoresis

• The polymerase chain reaction (PCR) can produce many copies of a specific target segment of DNA

• A three-step cycle brings about a chain reaction

  • Denature (heat to break the DNA strands)

  • Annealing (primers added to sequence)

  • Elongation (taq Polymerase replicates complementary strand)

• The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase