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Thread: Basic Conceptions in Genetics

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    Red face Basic Conceptions in Genetics

    Genome refers to a complete genetic sequence on one set of chromosomes of a organism. For example, the human genome is divided into 23 different chromosomes, including 22 autosomes and the X and Y sex chromosomes.

    Exons refer to the portion of genes that are eventually spliced together to form RNA.

    Introns refer to the spacing regions between the exons that are spliced out of precursor RNAs during RNA processing.

    Phenotype: An observed trait is referred to as a phenotype.

    Genotype: The genetic information defining the phenotype is called the genotype.

    Alleles: Alternative forms of a gene or a genetic marker are referred to as alleles.

    Haplotype: It refers to a group of alleles that are closely linked together at a genomic locus.
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    Point mutations: Mutations involving single nucleotides are refferred to as point mutations. Point mutations include transitions, transversions, missenses, and nonsense mutations.

    Transitions: Substitutions that a purine is replaced by another purine base, or a pyrimidine is replaced by another pyrimidine.

    Transversions: Substitution that a purine is replaced by a pyrimidine, or vice versa.

    Missense: The DNA sequence change occurs in a coding region and alters an amino acid.

    Nonsense: A nonsense mutation is a point mutation in a sequence of DNA that results in a premature stop codon.

    Important Concepts

    A gene product is usually a protein but can occasionally consist of RNA that is not translated, such as microRNAs.

    The transcription of genes is controlled primarily by transcription factors that bind to DNA sequences in the regulatory regions of genes. Also, gene expression is also influenced by epigenetic events, such as some DNA or histone modifications (inactivation, imprinting, methylation etc).

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    Last edited by Janis.Y.Chen; Fri 17th October '14 at 11:11pm.
    Clinical Pharmacy Specialist - Infectious Diseases

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    PharmD Year 1 TomHsiung's Avatar
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    Default Re: Basic Conceptions in Genetics

    Overview of Gene Expression

    As we have seen, producing a protein from information in a DNA gene is a two-step process. The first step is synthesis of an RNA that is complementary to one of the strands of DNA. This is called transcription. In the second step, called translation, the information in the RNA is used to make a polypeptide. Such an informational RNA is called a messenger RNA (mRNA) to denote the fact that it carries information - like a message - from a gene to the cell's protein factories.

    Like DNA and RNA, proteins are polymers - long, chain-like molecules. The monomers, or links, in the protein chain are called amino acids. DNA and protein have this informational relationship: Three nucleotides in the DNA gene stand for one amino acid in a protein.

    Figure 3.1 summarizes the process of expressing a protein-encoding gene and introduces the nomenclature we apply to the strands of DNA. Notice that the mRNA has the same sequence (except that U's substitute for T's) as the top strand (blue) of the DNA. An mRNA holds the information for making a polypeptide, so we say it "codes for" a polypeptide, or "encodes" a polypeptide. In this case, the mRNA codes for the following string of amino acids: methionine-serine-asparagine-alanine, which is abbreviated Met-Ser-Asn-Ala. We can see that the codeword (or codon) for methionine in this mRNA is the triplet AUG; similarly, the codons for serine, asparagine, and alanine are AGU, AAC, and GCG, respectively.

    Because the bottom DNA strand is complementary to the mRNA, we know that it served as the template for making the mRNA. Thus, we call the bottom strand the template strand, or the transcribed strand. For the same reason, the top strand is the nontemplate strand, or the non-transcribed strand. Because the top strand in our example has essentially the same coding properties as the corresponding mRNA, many geneticists call it the coding strand. The opposite strand would therefore be the anticoding strand. Also, since the top strand has the same sense as the mRNA, this same system of nomenclature refers to this top strand as the sense strand, and to the bottom strand as the antisense strand.

    Transcription
    As you might expect, transcription follows the same base-pairing rules as DNA replication: T, G, C, and A in the DNA pair with A, C, G, and U, respectively, in the RNA product. This base-pairing pattern ensures that an RNA transcript is a faithful copy of the gene.

    Of course, highly directed chemical reactions such as transcription do not happen at significant rates by themselves - they are enzyme catalyzed. The enzyme that directs transcription is called RNA polymerase. Transcription has three phases: initiation, elongation, and termination. The following is an outline of these three steps in bacteria:

    1) Initiation
    First, the enzyme recognizes a region called a promoter, which lies just "upstream" of the gene. The polymerase binds tightly to the promoter and causes localized melting, or separation, of the two DNA strands within the promoter. At least 12 bp are melted. Next, the polymerase starts building the RNA chain. The substrates, or building blocks, it uses for this job are four ribonucleoside triphosphate: ATP, GTP, CTP, and UTP. The first, or initiating, substrate is usually a purine nucleotide. After the first nucleotide is in place, the polymerase joins a second nucleotide to the first, forming the initial phosphodiester bond in RNA chain. Several nucleotides may be joined before the polymerase leaves the promoter and elongation begins.

    2) Elongation
    During the elongation phase of transcription, RNA polymerase directs the sequential binding of ribonucleotides to the growing RNA chain in the 5' --> 3' direction. As it does so, it moves along the DNA template, and the "bubble" of melted DNA moves with it. This melted region exposes the bases of the template DNA one by one so they can pair with the bases of the incoming ribonucleotides. As soon as the transcription machinery passes, the two DNA strands wind around each other again, re-forming the double helix. This points to two fundamental differences between transcription and DNA replication: a) RNA polymerase makes only on RNA strand during transcription, which means that it copies only one DNA strand in a given gene. (However, the opposite strand may be transcribed in another gene.) Transcription is therefore said to be asymmetrical. This contrasts with semiconservative DNA replication, in which both DNA strands are copied. b) In transcription, DNA melting is limited and transient. Only enough strand separation occurs to allow the polymerase to "read" the DNA template strand. However, during replication, the two parental DNA strands separate permanently.

    3) Termination
    Just as promoters serve as initiation signals for transcription, other regions at the ends of genes, called terminators, signal termination. These work in conjunction with RNA polymerase to loosen the association between RNA product and DNA template. The result is that the RNA dissociates from the RNA polymerase and DNA, thereby stopping transcription.
    Last edited by TomHsiung; Sun 25th December '16 at 3:00pm.
    B.S. Pharm, West China School of Pharmacy, Class of 2007, Health System Pharmacist, RPh. Hematology, Infectious Disease.

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    PharmD Year 1 TomHsiung's Avatar
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    Default Re: Basic Conceptions in Genetics

    Translation
    The mechanism of translation is also complex and fascinating.

    Transfer RNA: The Adapter Molecule
    The transcription mechanism was easy for molecular biologists to predict. RNA resembles DNA so closely that it follows the same base-pairing rules. By following these rules, RNA polymerase produces replicas of the genes it transcribes. But what rules govern the ribosome's translation of mRNA to protein?

    The adapter molecule in translation is indeed a small RNA that recognizes both RNA and amino acids; it is called transfer RNA (tRNA). tRNA has two "business ends". One end (the top of the model) attaches to an amino acid, which is amino acid-specific meaning only the specific amino acid could be attached to that specific tRNA(s) (degenerate). The attaching of amino acid to tRNA is catalyzed by aminoacyl-tRNA synthetase. The other end (the bottom of the model) contains a 3-bp sequence that pairs with a complementary 3-bp sequence in an mRNA. Such a triplet in mRNA is called a codon and its complement in a tRNA is called an anticodon.

    Initiation of Protein Synthesis
    We have just seen that three codons terminate translation (UAG, UAA, and UGA). A codon (AUG) also usually initiates translation. The mechanisms of these two processes are markedly different.

    We find AUG codons not only at the beginning of mRNAs, but also in the middle of messages. When they are at the beginning, AUGs serve as initiation codons, but when they are in the middle, they simply code for methionine. The initiation AUGs is distinguished from internal AUGs by a Shine-Dalgarno ribosome-binding sequence near the beginning of material mRNAs, and by a cap structure at the 5' end of eukaryotic mRNAs.

    Translation Elongation
    At the end of the initiation phase of translation, the initiating aminoacyl-tRNA is bound to a site on the ribosome called the P site. For elongation to occur, the ribosome needs to add amino acids one at a time to the initiating amino acid.

    Elongation begins with the binding of the second aminoacyl-tRNA to another site on the ribosome called the A site. This process requires an elongation factor called EF-Tu, where EF stands for "elongation factor," and energy provided by GTP.

    Next, a peptide bond must form between the two amino acids. The large ribosomal subunit contains an enzyme known as peptidyl transferase, which forms a peptide bond between the amino acid or peptide in the P site and the amino acid part of the aminoacyl tRNA in the A site. The result is a dipeptidyl-tRNA in the A site. The second amino of the dipeptide is still bound to its tRNA.

    The third step in elongation, translocation, involves the movement of the mRNA one codon's length through the ribosome. This maneuver transfers the dipeptidyl-tRNA from the A site to the P site and moves the deacetylated tRNA from the P site to another site, the E site, which provides an exit from the ribosome. Translocation requires another elongation factor called EF-G and GTP.

    Termination of Translation and mRNA Structure
    Three different codons cause termination of translation. Protein factors called release factors recognize these termination codons and cause translation to stop, with release of the polypeptide chain. The initiation codon at one end, and the termination codon at the other end of a coding region of a gene identify an open reading frame (ORF). It is called "open" because it contains no internal termination codons to interrupt the translation of the corresponding mRNA. The "reading frame" part of the name refers to the way the ribosome can read the mRNA in three different ways, or "frames," depending on where it starts.

    Basic Conceptions in Genetics-screen-shot-2016-12-25-at-3-54-30-pm-png
    Last edited by TomHsiung; Sun 25th December '16 at 3:55pm.
    B.S. Pharm, West China School of Pharmacy, Class of 2007, Health System Pharmacist, RPh. Hematology, Infectious Disease.

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    PharmD Year 1 TomHsiung's Avatar
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    Default Re: Basic Conceptions in Genetics

    Promoter Structure
    What is the special nature of a bacterial promoter that attracts RNA polymerase? David Pribnow compared several E. coli and phage promoters and discerned a region they held in common: a sequence of 6 or 7 bp centered approximately 10 bp upstream of the start of transcription. This was originally dubbed the "Pribnow box," but is now usually called the -10 box. Mark Ptashne and colleagues noticed another short sequence centered approximately 35 bp upstream of the transcription start site; it is known as the -35 box. Thousands of promoters have now been examined and a typical, or consensus sequence for each of these boxes has emerged.

    Basic Conceptions in Genetics-screen-shot-2016-12-25-at-9-10-02-pm-png

    These so-called consensus sequences represent probabilities. The capital letters in Figure 6.5 denote bases that have a high probability of being found in the given position. The lowercase letters correspond to bases that are usually found in the given position, but at a lower frequency than those denoted by capital letters. The probabilities are such that one rarely finds -10 or -35 boxes that match the consensus sequences perfectly. However, when such perfect matches are found, they tend to occur in very strong promoters that initiate transcription unusually actively. In fact, mutations that destroy matches with the consensus sequences tend to be down mutations. That is, they make the promoter weaker, resulting in less transcription. Mutations that make the promoter sequences more like the consensus sequences usually make the promoters stronger; these are called up mutations. The spacing between promoter elements is also important, and deletions or insertions that move the -10 and -35 boxes unnaturally close together or far apart are deleterious.

    Basic Conceptions in Genetics-screen-shot-2016-12-25-at-9-18-39-pm-png

    In addition to the -10 and -35 boxes, which we can call core promoter elements, some very strong promoters have an additional element farther upstream called an UP element. E. coli cells have seven genes (rrn genes) that encode rRNAs. Under rapid growth conditions, when rRNAs are required in abundance, these seven genes by themselves account for the majority of the transcription occurring in the cell. Obviously, the promoters driving these genes are extraordinarily powerful, and their UP elements are part of the explanation. Because the UP element is recognized by the polymerase itself, we conclude that it is a promoter element. Figure 6.6 shows the structure of one of these promoters, the rrnB P1 promoter. Upstream of the core promoter (blue), there is an UP element (red) between positions -40 and -60. We know that the UP element is a true promoter element because it stimulates transcription of the rrnB P1 gene by a factor of 30 in the presence of RNA polymerase alone.

    This promoter is also associated with three so-called Fis sites between positions -60 and -150, which are binding sites for the transcription-activator protein Fis. The Fis sites, because they do not bind RNA polymerase itself, are not classical promoter elements, but instead are members of another class of transcription-activating DNA elements called enhancers.

    The E. coli rrn promoters are also regulated by a pair small molecules: the initiating NTP (the iNTP) and an alarmone, guanosine 5'-diphosphate 3'-diphosphate (ppGpp). An abundance of iNTP indicates that the concentration of nucleotides is high, and therefore it is appropriate to synthesize plenty of rRNA. Accordingly, iNTP stabilizes the open promoter complex, stimulating transcription.

    On the other hand, when cells are starved for amino acids, protein synthesis cannot occur readily and the need for ribosomes (and rRNA) decreases. Ribosomes sense the lack of amino acids when uncharged tRNAs bind to the ribosomal site where aminoacyl-tRNA would normally bind. Under these conditions, a ribosome-associated protein called RelA receives the "alarm" and produces the "alarmone" ppGpp, which destabilizes open promoter complexes whose lifetimes are normally short, thus inhibiting transcription.

    The protein DskA also plays an important role. It binds to RNA polymerase and reduces the lifetimes of the rrn open promoters to a level at which they are responsive to changes in iNTP and ppGpp concentrations. Thus DskA is required for the regulation of rrn transcription by these two small molecules. Indeed, rrn transcription is insensitive to iNTP and ppGpp in mutants lacking DskA.

    Summary
    Bacterial promoters contain two regions centered approximately at -10 and -35 bp upstream of the transcription start site. In E. coli, these bear a greater or lesser resemblance to two consensus sequences: TATAAT and TTGACA, respectively. In general, the more closely reigons within a promoter resemble these consensus sequences, the stronger that promoter will be. Some extraordinarily strong promoters contain an extra element (an UP element) upstream of the core promoter. This makes these promoters even more attractive to RNA polymerase. Transcription from the rrn promoters responds positively to increases in the concentration of iNTP, and negatively to the alarmone ppGpp.
    Last edited by TomHsiung; Sun 25th December '16 at 9:43pm.
    B.S. Pharm, West China School of Pharmacy, Class of 2007, Health System Pharmacist, RPh. Hematology, Infectious Disease.

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    PharmD Year 1 TomHsiung's Avatar
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    Default Re: Basic Conceptions in Genetics

    Transcription Initiation
    Transcription initiation is complex. It is now commonly represented in four steps, as depicted in Figure 6.8: 1) formation of a closed promoter complex; 2) conversion of the closed promoter complex to an open promoter complex; 3) polymerizing the first few nucleotides (up to 10) while the polymerase remains at the promoter, in an initial transcribing complex; and 4) promoter clearance, in which the transcript becomes long enough to form a stable hybrid with the template strand. This helps to stabilize the transcription complex, and the polymerase changes to its elongation conformation and moves away from the promoter.

    Basic Conceptions in Genetics-screen-shot-2016-12-26-at-8-09-58-pm-png

    Sigma Stimulates Transcription Initiation
    Because σ directs tight binding of RNA polymerase to promoters, it places the enzyme in a position to initiate transcription - at the beginning of a gene. Therefore, we would expect σ to stimulate initiation of transcription. Results in some classic experiments suggests that σ enhanced both initiation and elongation. However, initiation is the rate-limiting step in transcription. Thus, σ could appear to stimulate elongation by stimulating initiation and thereby providing more initiated chains for core polymerase to elongate.

    Travers and Burgess proved that is the case by demonstrating that σ really does not accelerate the rate of RNA chain growth. They found that σ made no difference in the lengths of the RNAs in their experiment. Therefore, σ does not stimulate elongation, and the apparent stimulation in the previous experiment was simply an indirect effect of enhanced initiation.

    Summary
    Sigma stimulates initiation, but not elongation, of transcription.
    Last edited by TomHsiung; Mon 26th December '16 at 8:11pm.
    B.S. Pharm, West China School of Pharmacy, Class of 2007, Health System Pharmacist, RPh. Hematology, Infectious Disease.

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    PharmD Year 1 TomHsiung's Avatar
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    Default Re: Basic Conceptions in Genetics

    The Stochastic σ-Cyle Model
    The σ-cycle model that arose from Travers and Burgess's experiments called for the dissociation of σ from core as the polymerase undergoes promoter clearance and switches from initiation to elongation mode. This has come to be known as the obligate release version of the σ-cycle model. Although this model has held sway for 30 years and has considerable experimental support, it does not fit all the data at hand.

    Based on inconsistent evidences, an alternative view of the σ-cycle was proposed: the stochastic release model. This hypothesis holds that σ is indeed released from the core polymerase, but there is no discrete point during transcription at which this release is required; rather, it is released randomly. The preponderance of evidence now favors the stochastic release model.

    Studies confirmed that σ did indeed remain associated with the great majority (about 90%) of elongation complexes that had achieved promoter clearance (with transcripts 11 nt long). Again, this finding argued strongly against the obligate release model. But they also showed that about half of halted elongation complexes with longer transcripts had lost their σ-factors, in accord with the stochastic release model. Finally, their results suggested that some elongation complexes may retain their σ-factors throughout the transcription process. If that is true, these elongation complexes are avoiding the σ cycle altogether.
    Last edited by TomHsiung; Mon 26th December '16 at 9:00pm.
    B.S. Pharm, West China School of Pharmacy, Class of 2007, Health System Pharmacist, RPh. Hematology, Infectious Disease.

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