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BIOB12H3 (8)
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Chapter 11

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Biological Sciences
Dan Riggs

GENE EXPRESSION: FROM TRANSCRIPTION TO TRANSLATION 11.1 THE RELATIONSHIP BETWEEN GENES AND PROTEINS o Garrod (1908) exhibited that persons with rare inherited diseases were caused by the absence of specific enzymes  “inborn errors of metabolism”  Ex. alkaptonuria o Beadle and Tatum (1940s) studied Neurospoa , bread mold with few resources  assumed should be very sensitive to enzymatic deficiencies and easily detected o Irradiate mold and screen for mutations o Steps to experiment (Figure 11.1) 1. Spores irradiated to induce mutation 2. Allowed to grow in colonies in tubes containing a supplemented medium 3. Spores produced by colonies were tested to grow on original minimal medium  those failed are mutants  not to identify the mutant gene 4. Mutant cells were supplemented to grow in the minimal medium supplemented with vitamins but NOT amino acids  deficiency in an enzyme leading to a vitamin 5. The same cells in minimal medium supplemented with one or another vitamins shows the deficiency resides in a gene involved in the formation of pantothenic acid o A gene carries the information for the construction of a particular enzyme  “one gene – one enzyme” hypothesis  later became the “one gene – polypeptide” hypothesis o Ingram (1956) demonstrated a single mutation in a single gene has caused a single substitution in the amino acid sequence of a single polypeptide (Ex. sickle cell hemoglobin) o Transcription: the synthesis of an RNA from a DNA template o Overview of the flow of information in a eukaryotic cell (Figure 11.2) 1. Selected sites of DNA are transcribed into pre-mRNAs 2. Pre-mRNAs are processed into mRNAs 3. The mRNAs are transported out of the nucleus into the cytoplasm 4. mRNA is translated in polypeptides by ribosomes that move along the mRNA 5. Polypeptide folds to assume its native conformation o mRNA allows the cell the separate information storage from information utilization o Translation: a complex process that synthesizes proteins in the cytoplasm o Translation requires ribosomes: nonspecific components of the translation machinery o rRNA OR ribosomal RNAs are the RNAs of a ribosome, like mRNAs, each is transcribed from one of the DNA strands of a gene. They recognize and bind other molecules, provide structural support, and catalyze the chemical reaction in which amino acids are covalently linked to one another rd o tRNA OR transfer RNAs constitute a 3 major class of RNA that is required for protein synthesis. They are required to translate the information in the mRNA nucleotide code into the amino acid “alphabet” of a polypeptide o RNAs fold into a complex 3D shape, markedly different from one type of RNA to another; carry out diverse array of functions because of their different shapes o RNA folding is driven by the formation of regions having complementary base pairs 11.2 AN OVERIVIEW OF TRANSCRIPTION IN BOTH PROKARYOTIC AND EUKARYOTIC CELLS o Enzymes responsible for transcription in prokaryotic and eukaryotic cells are RNA polymerases, which incorporate nucleotides on at a time, into a strand of RNA whose sequence is complementary to one of the DNA strands which serves as a template o Steps in synthesis of an RNA 1. Association of the polymerase with the DNA template; site on DNA to which an RNA polymerase molecule binds prior to initiation transcription is the promoter (also contains information that determines which strand is transcribed where transcription begins)  with the help of transcription factors RNA polymerases are capable of recognizing promoters 2. Polymerase moves along the template DNA strand in a 3’  5’ direction (5’  3’ being synthesized in an antiparallel fashion) 3. As the polymerase progresses, the DNA is temporarily unwound, and the polymerase assembles a complementary strand of RNA that grows from its 5’ terminus in a 3’ direction 4. RNA polymerase catalyzes (RNA + NPPP  RNA + PP) n n+1 i - NPPPs = ribonucleoside triphosphate precursors (hydrolyzed into nucleoside monophosphates as they are polymerized into a covalent chain) - PP iecomes hydrolyzed to inorganic phosphate (P) whici releases large amount of free energy  making the incorporation of nucleotides essentially reversible 5. RNA moves down the DNA template and incorporates complementary nucleotides into the growing RNA chain (able to form a proper base pair with the DNA strand) 6. Once the polymerase has moved past a particular stretch of DNA, the DNA double helix reforms o RNA polymerases are capable of forming prodigiously long RNAs; the enzyme must remain attached to the DNA over long stretches of template (enzyme said to be processive)  also the enzyme must be associated loosely enough that it can move from nucleotide to nucleotide of the template o TRANSCRIPTION IN BACTERIA - Bacteria such a E.coli contain a single type of RNA polymerase of five subunits that are tightly associated to form a core enzyme; if purified and added to a solution of bacterial DNA and ribonucleoside triphosphates, the enzyme bids to the DNA and synthesizes RNA - Core enzyme attaches to random sites in DNA - If a purified accessory polypeptide, sigma factor, is added to the RNA polymerase before it attaches to DNA, transcription begins at selected locations  increases the enzyme’s affinity for promoter sites in DNA and decreases its affinity for DNA in general  binds to sequences at -10/-35 - Enzyme separates (or melts) the two DNA strands in the region surrounding the start site; makes the template strand accessible to the enzyme’s active site (resides at the back wall of the channel) - Once 10-12 nucleotides have been successfully incorporated into a growing transcript, the enzyme undergoes a conformational change into a transcriptional elongation complex that can move progressively along the DNA (sigma factor is released) o Proportions of the DNA preceding the initiation site (toward the 3’ end of the template) are said to be upstream from that site o Proportions of DNA succeeding it (toward the 5’ end of the template) are said to be downstream from that site o 35 bases upstream from the initiation site is the consensus sequence, TTGACA, indicates the most common version of the conserved sequence (some variation may occur) o Second conserved sequence is found ~ 10 bases upstream from the initiation site and occurs at the consensus sequence, TATAAT, named the TATA box/ Pribnow box after discoverer who identified where the precise nucleotide at which transcription begins o Rho, a ring-shaped protein is required for termination of bacterial transcription, or when it reaches a terminator sequence o TRANSCRIPTION AND RNA PROCESSING IN EUKARYOTIC CELLS - Eukaryotic cells have three distinct transcribing enzymes in the nucleus, each responsible for synthesizing a different group of RNAs - Recently discovered a fourth group RNA polymerase IV (plants only?)  siRNAS - Eukaryotic RNA polymerases can be distinguished by their susceptibility to α-amanitin Class Sensitivity to α-amanitin RNAS Pol I Insensitive Large (rRNAs) Pol II Very sensitive mRNAs Pol II Moderately sensitive Small (5s, tRNAs) - Major distinction between transcription in prokaryotes and eukaryotes is the requirement in eukaryotes for a large variety of accessory proteins (transcription factors)  bind polymerase to the DNA template, to the initiation of transcription, to its elongation and termination - 3 major types of RNAs (mRNAs, rRNAs, and tRNAs – are derived from precursor RNA molecules that are considerable longer than the final RNA product - RNAs are synthesized as precursors because the initial precursor is equivalent in length to the full length of the DNA transcribed called the primary transcript (pre-RNA)  corresponding segment of DNA from which a primary transcript is the transcription unit. Primary transcripts usually have a fleeting existence, being processed into smaller, functional RNAs but a series of “cut-and-paste” reactions 11.3 SYNTHESIS AND PROCESSING OF RIBOSOMAL AND TRANSFER RNAS o DNA sequences encoding rRNA are normally repeated hundreds of times, rDNA, is typically clustered in one or a few regions of the genome; human genome has 5 rDNA clusters, each on a different chromosome o In interphase (nondividing), the cell clusters of rDNA are gathered together as part of one or more irregularly shaped nuclear structures known as nucleoli (nucleolus-singular) which function in producing ribosomes o 49 ribosomal proteins make the 60S subunit of a ribosome o 33 ribsomal proteins make the 40S subunit of a ribosome o A ribosome = 60S + 40S = 80S o SYNTHESIZING THE rRNA PRECURSOR - Shorter fibrils are RNA molecules of fewer nucleotides that are attached to polymerase molecules bound to the DNA closer to the transcription initiation site - Longer the fibril, the closer the transcript to completion - RNA fibrils contain associated particles which consist of RNA and protein that work together to convert rRNA precursors to their final rRNA products and assemble them into ribosomal subunits - Region of the DNA fiber between adjacent transcription units is devoid of nascent RNA chains  this region of the ribosomal gene cluster is not transcribed it is known as the, nontranscribed spacer, which are present between various types of tandemly repeated genes including those of tRNA and histones - Transcription of rRNA genes by multiple polymerases in my eukaryotes, rRNA gene clusters (NORs) exists as tandem repeats on several chromosomes o PROCESSING THE rRNA PRECURSOR - S (Svedberg) value: correlated with size and shape, so larger S value means that the molecule is larger (perhaps a greater surface area) than one having a lower S value  S values are not additive (Ex. 28S + 18S + 5.8S /= 45) - RNA size can be estimated on sedimentation of the molecule during centrifugation - 4 distinct ribosomal RNAs: 3 in the large subunit and 1 in the small subunit - Large subunit: 28S, 5.8S, and 5S (from 45s broken down in 4 cuts  liberate parts of molecule - Small subunit: 18S - 28S, 18S, and 5.8S  carved by various nucleases from a single primary transcript (pre- rRNA)  stable rRNAs that assemble with proteins to build ribosomes - 5S is synthesized from a separate RNA precursor outside the nucleolus - Pre-rRNA nucleotides that were altered posttranscriptionally remain as part of the final product  unaltered sections are discarded during processing - Pulse chase experiment: 1. PULSE: add radioactive precursor to cells for short period. Get incorporation of label into macromolecule pool 2. CHASE: wash cells to remove radioactive precursor, then examine radioactive macromolecules after an incubation period 3. Utility: a basic biochemical tool to “tag” a molecule or population of molecules, and determine their fate over a time course - Analogy: dye in water flow i. 45S primary transcript is the first species to become labeled, seen as a peak of radioactivity in nucleolar RNA fraction after 10 minutes ii. After an hour, the 45S disappears from nucleolus and is replaced by 32S RNA (one of the two major products of 45S)  peaks from 40-150 minutes iii. 32S a precursor to the mature 28S and 5.8S rRNAs iv. Other product of 45S pre-rRA leaves the nucleolus quite rapidly and appears in the cytoplasm (40min) v. After 2 or more hours, nearly all the radioactivity has left the nucleolus and has accumulated as 28S and 18S rRNAs of the cytoplasm vi. In the 4S RNA peak includes the 5.8S rRNA and methyl groups that have been transferred to small tRNAs o Penman experiment - How is rRNA made and processed and where do these events occur? 1. Label cells for short period (pulse) 2. Wash away label. Start incubation (chase) 3. Prepare nucleolar and cytoplasmic fractions 4. Isolate RNA from these fractions 5. Separate the RNA by size (centrifugation) 6. Determine the distribution of RNA molecules and follow the fate of the initial radiolabelled molecules - 5. The basis of sedimentation/centrifugation (move based on size) poke hole, allow to drip, collect fractions  Then measure amount by plotting fraction # vs another parameter (might be absorbance value; optical density: OD 260nm, or it might be counts per minute (for radioactive labeling) - 6. 4 panels: i. Top: nucleolar fractions ii. Bottom: cytoplasmic factors iii. Blue lines: All RNA (absorbance measurement)  total RNA doesn’t change iv. Red lines: pulse labeled RNA (radioactivity measurement) v. Conclude: after 10’ chase most RNA* is 45S, and still in nucleolar fraction o Small, nucleolar RNAs (snoRNAs) required for mRNA processing that are small (90 – 300 nucleotides long) and that function in the nucleus  packaged with particular proteins to form small, nucleolar RNPs (snoRNPs) which play an important role in the maturation and assembly of ribosomal RNAs o 5S, ~120 nucleotides long, part of the large subunit  following synthesis, the 5S rRNA is transported to the nucleolus to join the other components involved in the assembly of the ribosomal units  5S rRNA genes are transcribed by RNA polymerase III (can bind to the promoter site located within the transcribed portion of the target gene o tRNAs are synthesized from genes that are found in small clusters scattered around the genome  a single cluster typically contains multiple copies of different tRNA genes, and conversely, the DNA sequence encoding a given tRNA is typically found in more than one cluster  transcribed by RNA polymerase III and the promoter lies in the coding section 11.4 SYNTHESIS AND PROCESSING OF MESSENGER RNAS o heterogeneous nuclear RNAs (hnRNAs) : a large group of RNA molecules that share the following properties: 1) they heave large molecular weights (up to 80S, or 50000 nucleotides); 2) they represent many different nucleotide sequences; and 3) they are found only in the nucleus  includes pre-mRNAs o Large rapidly labeled hnRNAs were primarily precursors to the smaller cytoplasmic mRNAs o ~30,000 active genes – many transcripts of various sizes (for any genes, large or small) o Pulse Chase Experiment - Blue lines, optical density  provide information about the amount of RNA in each of the following centrifugation  18S and 28S rRNA present - Red lines, radioactivity in each fraction, - label cells for short period of time with P - Purify RNA, centrifuge to separate molecules by size - Determine OD (RNA) and radioactivity (labeled RNA) - 30 min  newly synthesized are is large RNA (hnRNA) larger than 18S and 28S - 3 hours with actinomycin D (prevents further synthesis of RNA)  large hmRNA have been processed into smaller RNA products  ~90% of the total RNA is rRNA - RNA pattern does not change (red line a little but shifted to the right  smaller size mRNA) o Half lives of mRNA vary depending on the particular species  - ‘half-life’ of hnRNA/mRNA: minutes to hours (Avg 40 min) - ‘half-life’ of rRNAs: days/months; accumulation occurs o Expression control: - Nuclear gene transcription by RNA pol II  - Heterogeneous nuclear RNA (hnRNA)  processing  - Messenger RNA (mRNA)  particular ½ life: articulating and prolonging it - Export to cytoplasm - Translation (ribosomes) o THE MACHINERY FOR MRNA TRANSCRIPTION - All eukaryotic precursors are synthesized by RNA polymerase II, an enzyme composed of a dozen subunits - RNA polymerase II occurs in cooperation with a number of general transcription factors (GTFs) - Promoters for RNA polymerase II lie to the 5’ side of each transcription unit (transcription factor, sigma, brines core polymerase to the right site)  lies between 24 and 32 bases upstream for site of initiated transcription - In eukaryotes TATA box ~-25 - TATA box of the DNA is the site of assembly of the preinitiation complex that contains GTFs and polymerase  must assemble before transcription of the gene can be initiated o PREINITIATION COMPLEX - TATA-binding protein (TBP) recognizes the TATA box of eukaryotes  bends DNA and facilitates unwinding - TBP is present as a subunit of a much larger protein complex called TFIID which is also binded to TAFs (TBP-associated factors) - GTFs : TBP, TFIIA and TFIIB bound to promoter provides a platform for the subsequent binding of the huge, multisubunit RNA polymerase with its attached TFIIF - RNA polymerase is tethered until TFIIF acts ( a protein kinases: phosphorylates the carboxyl terminal domain of Pol II where 52 repeats of 7AAs exist)  2 other subunits function as DNA unwinding enzymes (helicases) - DNA helicase is required to separate DNA strands of the promoter, allowing polymerase access to the template strand - Once transcription begins, certain GTFs may left behind while others are released from the complex  as long as TFIID remains bound to the promoter, additional RNA polymerase molecules may be able to attach to the promoter site and initiate additional rounds of transcription without delay - Carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II has an unusual structure (7AAs – 52 repeats)  catalyzed by TFIIH - RNA polymerase engaged in elongation may be associated with a number of large accessory proteins o STUCTURE OF MRNAS - Contain a specific sequence of nucleotides encoding a specific polypeptide - Found in the cytoplasm - Attached to ribosomes when they are translated - Most contain a significant noncoding segment, that does not direct the assembly of amino acids  found at 5’ and 3’ ends of a mRNA and contain sequences that have important regulatory roles - Special modifications are their 5’ and 3; termini (not found on bacteria mRNA, tRNA, and rRNAs Eukaryotic mRNA has a poly A tail at the 3’ and a 5’ cap o SPLIT GENES: AN UNEXPECTED FINDING - A number of adenovirus’ had the same 150-200 nucleotide 5’ terminus  one might expect that this leader sequence represents a repeated stretch of nucleotides located near the promoter region of each of the genes for there mRNAs - The leader sequence is transcribed from three distinct and separate segments of DNA with DNA sequences inbetween - Intervening sequences: regions of DNA that are between coding sequences if a gene and that are therefore missing from corresponding mRNA - ~600 bases located directly within a part of the goblin gene that coded for the amino acid sequence of the globin polypeptide - Split genes are genes with intervening sequences - Exons: those parts of a split gene that contribute to a mature RNA product - Introns: those parts of a split gene that correspond to the intervening sequences  found in all types of genes, those that encode: tRNAs, rRNAs, and mRNAs - Possibility that cells produce a primary transcript where portions of RNA corresponding to intros are somehow removed  would explain why hnRNA is way larger than mRNA - A few pre-mRNAs have been determined - Single stranded DNA and RNA can also bind to another as long as their nucleotides are complemtary; basis of the technique of DNA-RNA hybridization o THE PROCESSING OF EUKARYOTIC MESSENGER RNAS - RNA polymerase II assembles a primary transcript that is complementary to the DNA of the entire transcription unit - RNA transcripts become associated with proteins and larger particles while they are still in the process of being synthesized; proteins and ribonucleoproteins include agents responsible for converting the primary transcript into a mature messenger - Requires addition of 5’ cap and 3’ poly{A) tail at the ends of the transcript, removal of intervening introns  once processing on the mRNP, which consists of mRNA and associated proteins  ready for the nucleus o 5’CAPS and 3’ POLY(A) TAILS - 5’ ends of all RNAs initially possess a triphosphate derived from the first nucleoside triphosphate incorporated at the site of initiation of RNA synthesis - Once the 5’ end of the mRNA precursor is synthesized, several enzyme activities act on this end of the molecule - 1) The last of the three phosphates is removed  5’ terminus now a diphosphate - 2) A GMP is added in an inverted orientation so that the 5’ end of the guanosine is facing the 5’ end of the RNA chain - 3) different methyltransferases add a methyl group to the
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