Genetics: Molecular Genetics

Chapters 11-16: Molecular Genetics

• DNA is the Genetic Material.

– RNA can be the genetic material of a virus.

– RNA often functions structurally or as an information intermediate.

• Nucleic Acid Structure (pg 238-255).

– Levels of structure:

• 1) Nucleotides (A, G, T or U and C) are the repeating subunits of DNA structure.

• 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:

– Pyrimidines: cytosine

• Nucleotides distinguish DNA from RNA.

– DNA deoxyribose versus RNA ribose sugar.

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

– Nucleotides are linked together to form a strand.

• 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; the sequence from the previous page TAACCG and 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.

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

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– 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).

– Hydrogen bonds (2 or 3) form between the bases of the complementary nucleotides.

– 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 strands also fold into secondary and tertiary structures.

• Primary structure similar to DNA except what previously noted.

– Several hundred or thousand nucleotides in length, shorter than chromosomal DNA.

• 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).

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– Ex: tRNA folds (2^o) and interacts with an amino acid (3^o).

• DNA Replication (Chapter. 11).

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

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

• Structural Overview.

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

– Strands are antiparallel (based on sugar-phosphate orientation) one strand runs 5’à3’ while the other runs 3’à5’.

– Basis of DNA replication is complementarity.

• Two new strands are made complementary to the 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 from one both of which has one old strand and one new strand this is semiconservative replication.

– Prokaryotic Replication.

• Much early research focused on replication in E. coli.

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– Bidirectional replication involves two replication forks moving in opposite directions outward from the origin of replication.

• The DnaA protein of bacteria binds a specific DNA sequence in the oriC and causes the DNA region around the site to denature and then recruits DNA helicase (DnaB helicase).

• DNA helicase uses energy to separate the strands of DNA by breaking hydrogen bonds between complementary strands while moving in a 5’à3’ direction.

– Topoisomerase travels ahead of DNA helicase to ease supercoiling.

– Single-strand binding protein binds the new single strands and prevents them from reforming a double helix.

– Then small RNA primers must be made (primase) these are made to allow DNA replication to begin.

– DNA polymerases link nucleotides to synthesize daughter strands.

• Three DNA polymerases polI and polIII are important for DNA replication while polII is involved in DNA repair.

– 1) Polymerases can only elongate strands need a primer to start from (RNA primer).

– 2) Polymerases can only attach nucleotides in the 5’à3’ direction.

• Synthesis of the two new daughter strands is done differently for each strand however each starts at an RNA primer(s).

• DNA pol. catalyzes the attachment of nucleotides to the 3’ end of the RNA primer in a 5’à3’ direction.

– In leading strand synthesis one primer is made and then DNA pol. can continuously attach nucleotides in a 5’à3’ direction (towards replication fork).

– In lagging strand synthesis DNA pol. makes small fragments of DNA (Okazaki fragments) attached to primers going away from the replication fork but still in a 5’à3’ direction.

» Each Okazaki fragment is ~ 1,000-2,000 base pairs (bacteria), small RNA primer + DNA sequences.

– DNA polymerase (polI) removes primers of the lagging strand adds in the DNA and DNA ligase links the fragments.

• Three steps:

• 1)

• 2) DNA is filled into the place of the RNA primer by polI.

• 3) This leaves one space between two nucleotides where a phosphodiester bond is formed by DNA ligase.

– Replication is terminated when the replication forks meet at the terminus sequences.

• The E. coli chromosome contains a pair of termination sequences (ter) which when bound by the ter-binding protein stop the replication forks.

• DNA ligase then joins the four DNA strands.

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– Topoisomerase breaks the strands and reforms them to make two separate chromosomes (genomes).

– Fidelity of DNA replication ensured by proofreading mechanisms.

• polIII only makes 1 error every 100,000,000 nucleotides this is due to complementary base pairing and the removal of mistakes by DNA polymerase itself.

– Exonuclease activity and proofreading function.

• Eukaryotic DNA Replication.

– The most information is known about prokaryotes, research has been done in eukaryotes particularly in yeast and mammal cell culture.

– DNA helicases, primases, polymerases, ligases, topoisomerases and single-strand binding proteins have been identified in eukaryotes.

– More complex than prokaryotic replication:

• Larger, linear, more in number and more tightly packed chromosomes (nucleosomes).

• More regulation of the cell cycle.

– Multiple origins of replication in a linear chromosome.

• Autonomous replicating sequences (ARS) are necessary for replication initiation and found in more than one spot in a linear chromosome.

– ARS sequence is (A or T)TTTAT(A or G)TTT(A or T).

– Several polymerases in eukaryotes each with different functions.

– Nucleosomes remain attached to one strand and are synthesized de novo to accommodate new DNA.

• The two daughter DNA helices have a mixture of old and new histones for nucleosome formation.

– Ends of eukaryotic chromosomes are replicated by telomerase enzyme.

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• Overview of Gene Expression (Chapter 12, pg. 321-335).

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

– How do we get from gene to protein?

• Gene expression: DNA à RNA à Protein; using the processes of transcription and translation.

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

– 2)

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

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

– Genes are organized into functional groups of base sequences.

• 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.

– Terminator sequence signals the end of transcription.

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

• Some sequences in the gene ensure that the mRNA will be correctly made into protein.

• These sequences function within the mRNA itself and are use for the translation process.

– There may be site which the ribosome can recognize and bind the mRNA (bacterial Shine-Delgarno sequence).

» The sequence is actually complementary to ribosomal RNA (rRNA), which is used as part of ribosomal structure.

– During synthesis of the protein the information held in the mRNA is read in three nucleotide steps called codons.

» This is followed by many more codons which dictate the sequence of amino acids to build the protein.

» A stop codon signals the end of translation (a codon that does not encode for a particular amino acid).

– Transcription: DNA information is accessed to produce a molecule of RNA.

• 1) RNA polymerase binds to the promoter sequence with the aid of many transcription factors.

– Factors help with recognition and loading onto the promoter.

• 2) DNA strand begins to unwind and one strand is used as a template for building the RNA.

– A_RNA-T_DNA, U_RNA-A_DNA, G_RNA-C_DNA and C_RNA-G_DNA.

• 3) RNA synthesis stops at the terminator sequence.

– Three stages of transcription:

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

– Eukaryotic Promoter sequence includes TATA box (determines precise transcription start site, -25 nucleotides) and CAAT box (bound by general transcription factors).

» TFIID binds first, to the TATA box and TFIIB binds TFIID and helps load RNA polymerase.

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• Elongation: RNA polymerase slides down the DNA within the open complex synthesizing RNA.

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

– Eukaryotic pre-mRNA is often modified before they are functional mRNA.

• 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.

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

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

» Believed some may contain transcription regulatory sequences.

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

– This makes the mature mRNA that can then leave the nucleus and find a ribosome.

– RNA Modification.

• Trimming: Cutting an RNA transcript into smaller pieces one or more of which may be functional (Ex: rRNA or tRNA).

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• Poly A tailing: Addition of a poly A tail (3’ end) important for RNA stability and translation.

– AAUAAA is a polyadenylation sequence; endonuclease cleaves the mRNA and poly A-polymerase recognizes the sequence and adds adenosine nucleotides.

• Splicing: Spliceosome made of proteins and small nuclear RNAs (snRNP) form around two exons surrounding one intron.

– This forms the lariat bringing both exons in close proximity so they can be attached to one another, this repeats for all exons of the transcript.

– Translation: Genetic code within the mRNA is used to generate polypeptides with specific amino acid sequences.

• 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.

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• 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.

• 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.

• The genetic code is universal for all organisms with very few exceptions (Table 12-3).

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

• In order for translation of the mRNA: ribosomes, tRNAs, protein factors and small molecules 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).

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• 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) is reached.

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

– Ribosome structure and assembly.

• 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).

– Three stages of translation: Initiation elongation and termination.

• Initiation: The mRNA, tRNA^met and ribosome are involved with translation initiation factors (IF1, IF2 and IF3 or eIF); loading of the small subunit occurs onto the Shine-Delgarno (5’-cap in eukaryotes) sequence of the mRNA, then loading of the tRNA^met into the P site and finally the large subunit.

• Elongation: Polypeptide synthesis occurs; a new tRNA brings in an amino acid to the A site to attach it to the growing chain with the peptidyltransferase enzyme.

– Elongation factor EF-Tu provides energy for tRNA binding and EF-G provides energy for translocation of the tRNA peptide complex.

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– Each termination codon (Ex: amber, AUG) is recognized by a release factor (RF1 or RF2 (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.

– The sequence of a protein determines its structure and function.

• The amino acid sequence of a protein is called its primary structure (1^o) and indicates further structure of the polypeptide and the function.

• Secondary structure (2^o) of proteins includes hydrogen bonding of the backbone of the amino acid chain forming a-helices or b-pleated sheets (specialized structures).

• Tertiary structure (3^o) is 3D folding of the protein, can be a functional unit at this point.

• Quaternary structure (4^o)

• Bacterial Gene Regulation (Chapter 15, pg 395-414).

– Most bacterial genomes consist of a few thousand genes on a circular chromosome.

– The term gene regulation means the level of expression can vary depending on the needs of the cell.

• There are genes that do not vary in their expression they are called constitutive genes.

• Many genes however are considered regulated, so that proteins are only made when that particular protein is needed.

– Helps eliminate wasting energy; costs energy to make proteins can also cost energy to break them down.

– Commonly regulated genetic pathways: Metabolism, environmental stress response and cell division.

• The expression of structural genes (part that encodes for a particular polypeptide) is a multistep process leading to the production of functional proteins.

– There are layers of regulation at every step: Transcriptional, translational and posttranslational regulation.

– Transcriptional Regulation.

• Genes can be either “on or off” meaning expression has increased or decreased.

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– These factors can be repressors (negative control) or activators (positive control).

– Furthermore, effector molecules can bind these repressors or activators to control their function.

– These effectors are classified by what they do:

» An inducer increases transcription by binding an activator (helping it) or repressor (keeping it from functioning).

» Corepressor can inhibit an activator (by binding it) or bind a repressor increasing its effectiveness.

– These effectors lend their names to transcriptional systems in bacteria: An inducible system or repressible system.

– Jacques Monod and Francois Jacob studied enzyme adaptation.

• This is the idea that enzymes for utilization of a particular substance are only made after exposure to that substance (lac operon).

– Bacterial cells increase production of lactose utilization enzymes up to10,000 fold when exposed to lactose.

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– The lac operon (inducible operon) encodes for proteins involved in lactose metabolism.

• Multiple structural genes under the transcriptional control of one promoter is an operon (common in bacteria not eukaryotes).

• Operons contain a promoter (start) and terminator sequence (stop) with two or more structural genes between them.

• To control the ability of RNA polymerase to function an operator (repressor binding) site is present.

• Specific to the lac operon is a CAP site (catabolite activator protein) for activator binding.

• The structural genes lacZ, lacY and lacA are used for lactose metabolism.

• The lacI gene encodes the repressor for this inducible operon; the lacI gene is constitutively expressed.

– So in normal conditions (no lactose present) lacI protein is expressed, it binds the operator, blocks RNA polymerase and the operon is in the “off position”.

• When lactose is present a form of the sugar (allolactose) can bind the repressor blocking its ability to bind the operator allowing RNA polymerase to bind the promoter and transcribe the structural genes.

• Further regulation results from increased CAP which results from low glucose (preferred sugar, energy, source).

– Low glucose leads to increased CAP and a small metabolite called cAMP which bind cooperatively to the CAP site and act as an activator (Fig. 15-6).

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• Operon encodes for enzymes needed for tryptophan biosynthesis (trpE, trpD, trpC, trpB and trpA).

• The trpR and trpL genes regulate this operon in two different ways.

– A repressor is encoded for by trpR, when tryptophan levels are low in the cell the trpR protein cannot bind the operator.

– These conditions lead to expression of the structural genes and more tryptophan being made.

– When tryptophan levels are high, tryptophan acts as a corepressor with the trp repressor.

» This stops expression of the structural genes.

– The trpL gene mediates attenuation.

» This leads to short pieces of mRNA being made but not passed the attenuator sequence so it includes no structural genes.

» This only occurs if there is an abundance of tRNA^trp.

– Anabolic pathways tend to be repressible operons while catabolic pathways tend to be inducible.

– Regulation can occur translationally and posttranslationally.

• Translational initiation can be inhibited by translational repressors which bind mRNA and inhibit the process (more common than translational activators).

– Could bind near the Shine-Delgarno sequence.

• A common form of posttranslational feedback is negative feedback inhibition (bacteria and eukaryotes).

– The product of the pathway comes back and inhibits an important step in the pathway.

• Eukaryotic gene regulation (Chapter 16).

– More complex cell structure and multicellularity demand more (and more complex) gene regulation.

– All (almost) cells in an organism contain the same genetic material yet the cell looks and provides a different function (Ex: nerve and skin cell).

• The difference is provided by the regulation (on or off) of certain cell specific genes.

– The molecular mechanisms of regulation in eukaryotic cells are similar to prokaryotes.

– There are new levels of regulation along with transcriptional, translational and posttranslational; including genome and RNA processing steps of regulation.

– Regulation of DNA and Chromatin Structure.

• Gene amplification, gene rearrangement, DNA methylation and chromatin conformation can alter gene expression.

– Gene amplification is used only occasionally and results in an increased copy number of that gene in the chromosome (tandem repeats) or minichromosomes (circular and separate).

– Gene rearrangement occurs when genes are moved to make a new arrangement in response to environmental stimuli (Ex: antibody production).

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– Chromatin itself can be wound more tightly (closed conformation) or less tightly (open conformation) changing the ability of transcription factors to access genes in that area.

– Transcription Regulation.

• Transcription factors are proteins that influence the transcription of a given gene, two types are basal or regulatory transcription factors.

– Basal are general factors needed for transcription of any gene.

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• Regulatory factors commonly recognize and bind to specific DNA sequences.

– These DNA sequences are known as response or control elements (specific DNA sequence for each factor).

– Regulatory factors can act as activators or repressors.

» An activator may act to help load the general factors needed for transcription initiation, while a repressor may block recruitment of the general factors.

• Regulatory factors have different domains (areas of function).

– One could be specifically for interacting with DNA while another domain may interact with another protein (perhaps a general transcription factor).

– Transcription factors often form dimers (homo- or hetero-).

– Transcription factors recognize response elements that can up- or down-regulate transcription.

• An enhancer is a response element that will bind a transcription factor and stimulate (up-regulate) transcription.

• An silencer is a response element that will bind a transcription factor and inhibit (down-regulate) transcription.

– Function of regulatory transcription factors can be modulated in three ways.

• 1) Binding of an effector molecule (Ex: hormone, second messenger or other protein).

• 2)

• 3) Covalent modification (phosphorylation).

• Regulation of RNA processing and Translation.

– RNA processing can be regulated at the level of trimming, splicing, capping, poly A tailing and RNA editing.

– Concentration of mRNA will also change expression.

– Controlling the ability of the mRNA to be translated is another way to regulate gene expression.

– Alternative splicing.

• Different exons may be used or unused in making mRNA allowing one gene to form more than just one protein.

– Stability of mRNA changes mRNA concentration and can influence gene expression.

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– Poly A-binding protein (PABP) recognizes this tail, exonucleases shorten this tail and eventually the PABP cannot bind this signals the mRNA for degradation.

– The 3’ untranslated region (3’-UTR) can have an AU-rich element (ARE) this usually is found in short lived mRNA.

– Phosphorylation of translation initiation factors can alter translation rate.

• Phosphorylation of eIF2 inhibits translation while phosphorylation of eIF4F increases the translation rate.