Chapter 16. Molecular Basis for Inheritance and Chapter 17. Gene to Protein

• CH. 16: The Molecular Basis for Inheritance.

– DNA as the Genetic Material.

• The search for genetic material led to DNA.

• Watson and Crick Discovered the double Helix by building models to confirm X-ray data.

– DNA Replication and Repair.

• During DNA Replication base pairing enables existing DNA strands to serve as templates for new complementary strands.

• A large team of enzymes and other proteins carries out DNA replication.

• Enzymes proofread DNA during its replication and repair damage in existing DNA.

• A special mechanism is used to replicate the ends of DNA.

• CH. 17: From Gene to Protein.

– The Connection between Genes and Proteins

• The study of metabolic defects provided evidence that genes specify proteins.

• Transcription and translation overview.

• Nucleotide triplets specify amino acids.

• Evolved early in the history of life.

– The Synthesis and Processing of RNA.

• Transcription is the DNA-directed synthesis of RNA.

• Eukaryotic cells modify RNA after transcription.

– The Synthesis of Protein.

• Translation is the RNA-directed synthesis of a protein.

• RNA plays multiple roles in the cell.

• Comparing prokaryotic and eukaryotic translation.

• Point mutations can affect protein structure and function.

• DNA is the Genetic Material.

– RNA can be the genetic material of a virus and can often functions structurally or as an information intermediate.

• Nucleic Acid Structure.

– Levels of structure:

• 1)

• 2) Nucleotides are linked together to form a strand of DNA or RNA this linear sequence is the primary structure.

• 3) Secondary structure is the formation of the double helix; two complementary strands wound around one another.

• 4) the double helix is bent around itself and proteins to form the tertiary structure (chromosomes or chromatids).

– Nucleotides the building blocks (subunits) of nucleic acids.

• Three components make up a nucleotide: sugar, phosphate and nitrogenous base.

• Two categories of nucleotides: purines and pyrimidines.

– Purines: adenine (A) and guanine (G) (double-ring base structure).

– Pyrimidines: cytosine (C), thymine (T) and uracil (U) (single-ring base structure).

• Nucleotides distinguish DNA from RNA.

–

– Thymine used in DNA, while Uracil is used in RNA.

– Watson and Crick discovered the double helix by building models to confirm X-ray data (Fig 16.5).

• Phosphates and sugars make the backbone of DNA or RNA and the phosphate group is connecting the sugar of one nucleotide to the sugar of the next nucleotide, this is a phosphodiester bond.

• DNA has direction and sequence specificity; for example the sequence TAACCG has the direction 5’ à 3’ (5’-TAACCG-3’).

• These bases cannot be shuffled and maintain there position this is what can give individual genes their specific sequences.

– DNA has a double helical structure (Fig 11.6 & 11.7).

• Watson and Crick proposed this double helical structure with the help of findings from Wilkins, Franklin and Chargaff.

• The two strands of DNA are antiparallel and complementary.

– Meaning each strand goes in an opposite direction (one strand 5à3 other 3à5) and that individual base pairs have a complement on the other strand.

» Adenine base pairs with Thymine (A-T) and Guanine with cytosine (G-C) (Fig. 16.6).

–

– There are ten bases per 360^o turn of DNA.

– Major and minor groves along DNA.

– Additional folding and proteins needed for chromatin or chromosome structure.

• RNA can also form complex structures.

– RNA is single stranded which allows for interactions between base pairs within a strand or between strands this can give RNA secondary structures like: Bulges, internal loops, multibranched junctions and stem loops (hairpins).

• DNA Replication and Repair.

– DNA replication is the copying of the genetic material of a cell.

– Meselson and Stahl were the first to identify replication as semiconservative (Fig 16.8).

• The original DNA strands are used for making the new strands to make two complete sets of DNA (chromosomes).

– During DNA replication, base pairing enables existing DNA strands to serve as templates for new complementary strands.

• The DNA only fits together because A bonds with T and G bonds with C (must retain complementation).

•

– Two new strands are made complementary to each of the old strands.

• The original DNA strand actually comes apart and is used as the template for building the new strands.

– This makes two double helices both of which have one old strand and one new strand this is semiconservative replication.

– A large team of enzymes carries out DNA replication.

• Much early research on replication focused on replication in the bacteria E. coli so we know more about it than we do about eukaryotic DNA replication.

• Bacterial chromosomes contain a single origin of replication (Ori or ARS, autonomous replication sequence) and replication proceeds bidirectionally (Fig 16.10) eukaryotic chromosomes have multiple origins.

– Bidirectional replication involves two replication forks moving in opposite directions outward from the origin of replication, this also happens in eukaryotic cells.

– Proteins are involved in recognizing the origin and opening the DNA strands for replication.

–

• DNA polymerases are the enzymes responsible for generating the new DNA strands by adding nucleotides one at a time.

– Polymerases can not add nucleotides de novo they must add new nucleotides onto a free 3’ –OH (Fig. 16.11).

– This fact and the antiparallel nature of DNA results in both template strands being replicated differently.

» One strand may be replicated continuously (leading strand) while the other is synthesized in small fragments (lagging strand) (Fig 16.13).

– These fragments are called Okazaki fragments and are only about 100-200 bases in eukaryotes.

» These short segments each must be attached to one another in order to make a complete DNA strand, DNA ligase is the enzyme responsible for joining the sugar-phosphate backbones of these fragments.

• DNA Polymerases can only elongate strands from the 3’-OH so they need a primer to start from (RNA primer).

– Primase is the enzyme that provides an RNA primer for the leading strand and primers for the lagging strand (Fig 16.14).

– Primers are removed replaced with DNA and ligated together.

»

• Replication machinery does not move it reels in parent DNA and extrudes new daughter DNA (Fig 16.16).

– Enzymes proofread DNA during its replication.

• DNA polymerase makes errors about once every 10,000 base pairs, however it proofreads every nucleotide and can correct these errors.

• When an error slips through special enzymes can carry out nucleotide excision repair.

–

– Ex: Thymine cross-linking due to UV exposure from the sun (Fig 16.17).

– The ends of DNA molecules are replicated by a special mechanism (Fig. 16.19).

• A primer at each end of the chromosome (for replication) leaves a blank space with no way to fill in the DNA.

–

• Telomerase is a special enzyme that lengthens the ends of the chromosomes (telomeres).

– This slows the rate of telomere shortening because the lengthened telomere can then be used for primer placement.

– May be an important cancer drug target.

• From Gene to Protein (Chapter 17).

– We know that genes hold the hereditary information encoding for proteins that give cells and organisms their traits.

– The connection between genes and proteins.

• Gene expression: DNA à RNA à Protein (Central Dogma); uses the processes: transcription and translation.

– Transcription and Translation are the two main processes linking gene to protein (overview) (Fig 17.12).

• 1) The first stage involves copying the gene sequence (DNA) into messenger RNA (mRNA) this is transcription.

– pre-mRNA must be processed to mature mRNA.

• 2)

• 3) The ribosome uses the mRNA, ribosome and transfer RNA (tRNA) to build proteins one amino acid at a time (translation).

• The tRNA recognizes sites on the mRNA and brings in the corresponding amino acids.

– In the genetic code nucleotide triplets specify amino acids.

• The genetic code allows for mRNA to be translated into a specific amino acid sequence.

• The mRNA is read in three nucleotide chunks called codons, each of which encodes for a specific amino acid (Fig. 17.13).

– Some codons indicate the start (AUG)or termination of translation (UGA).

• Since there are four nucleotides (A, G, U and C) and three nucleotides per codon there are 64 (4³) different codons.

• Since there are only 20 amino acids some codons are used more than once for a specific amino acid, this is the degenerate code (Fig. 17.4).

• The third base of a codon is sometimes called the wobble base since it may differ but still encode for the same amino acid.

– Ex: codon GGU, GGC, GGA and GGG encode for the amino acid glycine.

– Genetic code must have evolved early in the history of life.

•

• Ex: UGA is stop except in mammalian mitochondria where it encodes for tryptophan.

• The synthesis and processing of RNA.

– Transcription is the DNA-directed synthesis of RNA.

• Gene at the molecular level is a relatively short segment of DNA located within a larger chromosome.

• A gene has many important sections to it that play different roles during gene expression.

– Promoter sequence provides a signal for the start of transcription.

– Regulatory sequences are important because they bind proteins that control the speed of the genes expression.

–

– The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit.

• Three stages of transcription (Fig. 17.6):

– Initiation: The promoter sequence binds general transcription factors (Ex: TFIID & TFIIB) in order to load the RNA polymerase holoenzyme (Fig 17.7).

» TATA Box in promoter is bound by TFIID, this is the start of transcription

– Elongation: RNA polymerase slides down the DNA within the open complex synthesizing RNA (Fig 17.6).

– Termination: RNA polymerase reaches termination signal and it and RNA transcript fall off.

»

• Alteration of mRNA ends (Fig. 17.8).

– Unlike prokaryotic mRNA in eukaryotes RNA must be matured into mRNA.

– A cap (7-methyl guanosine) is placed on the 5’ end and poly-A tail on the 3’ end.

» A cap on the mRNA acts as a recognition site for the ribosome.

» The poly A tail adds stability to the mRNA.

• Split genes and RNA splicing.

– Another important modification is splicing which includes removing certain pieces of RNA and covalently linking the remaining pieces together.

» The pieces that are spliced out are called introns, these are noncoding regions.

» Believed some may contain transcription regulatory sequences and allows for one gene to become more than one protein.

– Exons are spliced back together and hold the information coding for protein synthesis.

» This makes the mature mRNA, that leaves the nucleus to find a ribosome.

– (Fig 17.11).

• The Synthesis of Protein (Fig 17.12).

– Translation is the RNA directed synthesis of a polypeptide.

• In order for translation of mRNA: ribosomes, tRNAs, protein factors and small molecules (energy) are also necessary.

• In eukaryotes the ribosome binds to the mRNA and scans for a start codon (often AUG, encodes for the a.a methionine).

• The ribosome slides in a 5’ à 3’ direction on the mRNA.

• As a codon is read the corresponding tRNA with the complementary sequence to the codon (anticodon) brings in the specific amino acid for that codon.

• This continues until the stop codon (Ex:UAA, UAG or UGA) is reached.

• This signals the end of translation and then the complex (ribosome, tRNAs and mRNA) dissociates from the polypeptide.

• Ribosomes have two subunits, large (50S or 60S) and small (30S or 40S), made of proteins and ribosomal RNA (rRNA).

– Ribosomes have three important sites: Aminoacyl site (A), peptidyl site (P) and exit site (E) (Fig. 17.15).

– Three stages of translation: Initiation, elongation and termination.

• Initiation: The mRNA, tRNA^met and ribosome are involved with translation initiation factors; loading of the small subunit occurs onto (5’-cap in eukaryotes or Shine-Delgarno sequence of prokaryotes) the mRNA, then loading of the tRNA^met into the P site and finally the large subunit (Fig 17.7).

• Elongation: Polypeptide synthesis occurs; a new tRNA brings in an amino acid to the A site and attaches it to the growing chain with the help of an enzyme (Fig. 17.18).

– The complex in the A site is then translocated into the P site while the empty tRNA is ejected from the E site.

»

• Termination: Occurs when a termination codon is reached.

– Each termination codon is recognized by a release factor (RF1 or RF2 in prokaryotes (eRF in eukaryotes)).

– When a termination codon is located in the A site The RF will bind this site releasing the finished primary protein, mRNA and tRNA (Fig. 17.19).

• Polyribosomes are strings of ribosomes on a single mRNA and allow for quick translation of an mRNA (Fig 17.20).

• From polypeptide to functional protein.

– The amino acid sequence of a protein or its primary structure indicates all further structure of the polypeptide and the function.

» This includes secondary and tertiary structures and even the proteins ability to interact with other proteins (quaternary structure).

– Point mutations can affect protein structure and function.

• Point mutations are chemical changes in a single base pair of a gene.

• If a mutation happens in germ cells or gametes then it may be passed on to offspring.

• If the mutation has an adverse effect then it is a genetic disorder.

– Ex: Sickle-cell anemia is due to a single nucleotide change leading to a single amino acid change in the protein hemoglobin (Fig 17.23).

• Types of point mutations (Fig 17.24):

– Base-pair Substitutions are the replacement of one nucleotide and its partner.

» Can lead to missense (changes codon to encode for a new amino acid) or nonesense (introduces a stop codon) mutations.

– Insertions or deletions add in extra bases or remove bases changing the codons.

» Alters the reading frame during translation, frameshift mutation.