Test 3 review - Prof. Mowsh PDF

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Lecture 12  All DNA polymerases catalyze addition to 3' end of a pre-existing nucleotide chain. One XMP is added to the 3' end of the growing chain, using dXTP and releasing PPi.  DNA polymerases can have one to two catalytic activities (in addition to acting as polymerases) o Some DNA polymerases have 5' to 3' exonuclease: Can remove nucleotides from 5' end of primer by hydrolysis. (Note that RNA can be hydrolyzed by the 5' to 3' exonuclease activity of DNA polymerase. ) o Most DNA polymerases have 3' to 5' exonuclease: Can remove nucleotides from 3' end of growing chain by hydrolysis. This allows the enzyme to proofread -- to 'back up' and remove nucleotides that were added in error by hydrolyzing the phosphodiester bonds it has just made (if the wrong base was put in). When it backs up, DNA pol. catalyzes the following reaction:  rxn A: chain (n+1 units long) + H2O ↔ chain (n units long) + XMP o The 3' to 5' exonuclease reaction is not the same as the reverse of the polymerization reaction  Here is the normal elongation reaction catalyzed by DNA polymerase (to the right):  rxn B: Chain (n units long) + XTP ↔ Chain (n+1 units long) +PPi  PCR o needs a primer to start DNA synthesis o In a living cell (in vivo) primase -- a type of RNA polymerase -- makes the necessary RNA primer. Then DNA polymerase can take over, adding on to the 3' end of the primer. o In a test tube (in vitro) you can omit primase and use an oligonucleotide (short polynucleotide, usually DNA) as primer (= prefab DNA primer) to force replication to begin wherever you want. o different from regular DNA synthesis:  no replication fork/discontinuous synthesis  Preformed DNA primer. Primase is absent, so no RNA primers are made. Oligonucleotides of DNA (not RNA) are added instead to act as primers.  Special Polymerase. The DNA polymerase used in this procedure is a special heat-resistant one (called Taq polymerase) that is not denatured when the temperature is raised to separate the two strands of the DNA. This special polymerase was isolated from bacteria that live in a hot spring.

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RNA o Types. There are 3 major types of RNA involved in translation: messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA).  mRNA to act as template -- determines order of amino acids  tRNA to carry the amino acids to the template, and line them up  rRNA (in ribosomes) to align the tRNA's carrying the amino acids and hook the amino acids together

Lecture 13  DNA polymerase binds to start signals for replication called origins, while RNA polymerase binds to start signals for transcription called promoters  One Strand is Template for RNA polymerase. For any one gene or region, RNA polymerase uses Crick or Watson, but not both, as template. RNA that is made is complementary (and antiparallel)

to the template strand. Note that an entire strand is not used as template throughout. The "Watson" strand of DNA is used as template in some sections and the "Crick" strand in others.  transcribed strand of DNA: template/antisense, complementary to RNA made o meanwhile, not transcribed strand is the sense strand/coding strand (identical to RNA that is made except the RNA has U instead of T)  usual RNA transcribed from the DNA is said to be "sense"  promoter details: o Promoters determine the direction of transcription. Promoter and enzyme are asymmetric; therefore once enzyme binds, the catalytic end of RNA pol. is "facing" in one direction, and that determines the direction of transcription (and therefore which strand will be template). o The promoter will be a double stranded sequence at the end of the gene where RNA polymerase starts (= on 3' end of template strand = on 5' end of sense strand). Going along the sense strand, the way the gene is usually written (5' to 3', left to right) the promoter is "upstream" of the gene (that is, upstream of the protein coding part).  more promoters than origins in prokaryotic DNA (need one P per mRNA made)  special sequences called terminators cause the end of transcription for RNA o stop signals for translation (stop codons) are different than terminators  process of initiation/termination of macromolecular synthesis is always more complex than chain growth (elongation)  DNA polymerase can proof read, but RNA poly probably d/n o generally assumed that RNA pol. does not need to proofread, because RNA molecules are working copies that can tolerate a few errors (and can be replaced by new copies transcribed from the DNA) o can assume you don't have to consider RNA proofreading when solving problems  DNA poly proofreads/cannot start new chains, RNA poly d/n proofread and can start new chains o no primer required in RNA synthesis, since RNA poly can start its own chains  messenger RNA must be single stranded to fit in a ribosome and be translated, so that's why you use one strand in any region (can't have both sense/anti-sense complementary RNA)  REVIEW ANTISENSE MRNA  converting dsRNA into antisense o (1) Some enzymes cut up long ds RNA into short ds pieces, called short interfering RNA (siRNA). o (2) Other enzymes degrade the 'sense' strand of the short ds RNA  RNAi (RNA interference) - when a short piece of antisense RNA hybridizes to mRNA and blocks translation, and/or triggers degradation of the mRNA by cell enzymes  microRNAs - when double stranded RNA is made by a single strand doubling back on itself to form a small hairpin which is then cut up by enzymes  It is easier and more effective to block translation with RNAi (short ds RNA) than with antisense RNA (longer, ss RNA).  The product of transcription does not have to be an mRNA -- it can be a tRNA, rRNA, microRNA, etc. RNA is NOT used as template to make more RNA TRANSLATION  mRNA read in triplets (codons) going 5' to 3'  translation starts at an AUG and ends when it reaches the first stop codon after that  Leaders & Trailers. The region before the first AUG is not translated. It is called a leader, or 5'UTR (un-translated-region) or 5'UTS (un-translated sequence). Translation generally stops before the

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end of the mRNA (at a stop codon -- UAG, UAA or UGA). The untranslated region after the stop codon is called a trailer, or 3' UTR or 3' UTS. three possible reading frames Transfer RNA (tRNA) = adaptor. Cell uses tRNA to match the codon in the mRNA (say AUG or CUA) with the corresponding amino acid (met or leu, respectively). Loading Enzymes. Adaptor must carry the correct amino acid. Cell uses loading enzymes to put the correct amino acids on to their respective tRNA's Actual number of dif. tRNA's is more than 20 (#of dif. amino acids) and less than 64 (# of dif. codons)

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Important reminder: The code table lists the codons, NOT the anticodons. The anticodon in the tRNA is the complement of the triplet shown in the table.

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how does the new peptide chain grow? o chain adds to newest AA  Catalyst for formation of peptide bonds is called peptidyl transferase because the growing peptide chain is transferred as described above. This catalyst is part of the ribosome. o peptide chain grows amino -> carboxyl  This follows because the amino acids are held down (attached to tRNA) by their COOH ends. So if chain must add to free end of next AA, must add to amino end of next AA. energy for peptide synthesis

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energy derived from splitting the tRNA~chain bond drives peptide bond synthesis (is a high energy bond) stops o peptide chain stops growing when the translation machine comes to a stop codon  no tRNAs for the stop codons, so there is no way that the chain can keep growing if a stop codon comes next ribosomes contain both RNAs and proteins o rRNA =/= ribosomes; rRNA is inside the ribosome o 1 site or groove for mRNA o 2 sites for loaded tRNA (called A and P); these sites bind both mRNA and (loaded) tRNA  A site binds amino acyl tRNA  P site binds peptidyl tRNA o 1 site for unloaded tRNA; called E for exit site b/c it binds empty, used tRNA before it is bumped off the ribosome; binds tRNA but not mRNA o The empty tRNA moves into the E site, The peptidyl tRNA moves into the P site, and The A site becomes empty, ready for the next AA-tRNA. o all ribosomes in an organism are the same o peptidyl transferase is part of the ribosome o ribosome moves down mRNA 5' to 3' (or mRNA slides through ribosome) as peptide is made amino to carboxyl translocation o (1) Messenger RNA & tRNA do not move relative to each other, but are pulled together. o (2) Messenger RNA (& tRNA's) move relative to the Ribosome. As translocation occurs, which part actually moves? Ribosome or mRNA?

Lecture 14  transcription and translation are usually coupled in prokaryotes but not in eukaryotes  protein synthesis uses up a lot of energy - Movement and binding tRNA both require energy which we are ignoring. You probably need at least 5 P's split from ATP (or GTP)* per AA added if you count all the steps involved, not just growth of peptide chain  attachment of individual ribosomes o when not in use, they come apart into subunits o when translation starts, subunits come together (one small subunit and one large subunit form a ribosome that clamps onto the mRNA/begins translation) o when translation ends, the two subunits come apart and return to the pool  polysomes - more than one ribosome can read a single message at one time o first ribosome attaches near the 5' end of the mRNA o ribosome moves down the mRNA toward the 3' end, making protein o 2nd ribosome attaches once the first ribosome has moved far enough down; 2nd attaches behind it on the 5' side and follows the first down the message o more ribosomes attach until the entire mRNA is covered with ribosomes o a polysome forms; mRNA covered with multiple ribosomes (polyribosome, polysome for short)  Each subunit is a ribonucleoprotein or RNP -- each subunit is made of at least one kind of rRNA and many proteins  Use of S values. Whole ribosomes, subunits of the ribosome, and different ribosomal RNA's, are identified by their sedimentation constants (S values) in an ultracentrifuge.

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Sizes in eukaryotes and prokaryotes are slightly different. There are two values (of S) given for the sizes of the RNA's and subunits -- the smaller number is for prokaryotes; the larger # for eukaryotes. Self assembly -- How does ribosome structure form? The structure of each subunit is determined by the primary sequences of the rRNA's and proteins in it. Just as a protein folds up into the most stable (lowest energy) 3D conformation, so rRNA + proteins of each subunit fold into a ribonucleoprotein particle or RNP with proper 3D shape and function. rRNA vs ribosomes. Be careful not to confuse ribosomes (containing both rRNA and protein) with ribosomal RNA peptidyl transferase o catalyzes formation of the peptide bond, and is part of the large subunit of the ribosome o The catalytic activity is a property of the rRNA in the large subunit, not a protein, so this is not really an enzyme (catalyst made of protein) but a ribozyme (catalyst made of RNA) o not the only ribozyme How does protein synthesis start? -- you need a special met-tRNA for the P site. (AUG used for both start and methionine) o Both tRNA's for met recognize the same codon and carry the same amino acid, but one fits only in the P site and one only in the A site Met is usually removed from the amino end of the protein before the peptide chain folds up. Start codons vs Promoters -- these are not the same. Start codons affect translation and promoters affect transcription STOPS o no stop tRNA (ribosome stalls when it comes to a stop codon, tRNA w/ a peptide chain in the P site, but no TRNA to fit in the A site) o protein called a release factor binds to the stop codon in the A site and triggers release of the completed peptide chain o once the peptide is released from its tRNA, the tRNA falls off the ribosome and the ribosome disassociates into subunits which fall off the mRNA any code w/ synonyms is said to be degenerate (e.g., UUU/C is phe) What is wobble?The same tRNA could be used for two or more synonomous codons if you allow a little flexibility in pairing between the base at position 3 of the codon and its corresponding base (position 1) in the anticodon. This flexibility in pairing is known as "wobble." o *Wobble = Several different codons are read by one tRNA, not several different tRNA's are used for the same codon o position 3 of the codon and position 1 of the anticodon are known as "the wobble position" in their respective codons/anticodons o By wobble, we mean that the two bases involved in pairing (the ones in the wobble position of the codon and anticodon respectively) can twist slightly relative to each other. Because of this flexibility of alignment, H bonds are possible between groups that don't normally base pair in totally double stranded nucleic acids. (Nucleotides in ds DNA or DNA/RNA linear hybrids cannot twist.)

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 I = A without it's amino (has C=O instead of C-NH2 at position 6) ~30 diff tRNAs it's uphill from AA → chain, but downhill from AA-tRNA → chain. 2 P are split off ATP to get the AA onto the tRNA, and it's a downhill slide from there (AA-tRNA) to make the chain. Note that a tRNA-AA connection is broken to add an amino acid, but the connection that is broken is between the last amino acid in the peptide chain and the tRNA, not between the new AA and its tRNA. o It is important to realize that the change in free energy is downhill for one reaction (splitting ATP) and uphill for the other (adding phosphates to XMPs).

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charging/loading reaction is very complex, but serves 2 functions: o increases accuracy/specificity

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o energy: overall rxn hooks ATP hydrolysis to protein synthesis enzyme is called loading enzyme, activating/charging enzyme or aminoacyl-tRNA synthetase (different names emphasize different jobs of enzyme) o loading enzyme -- ferries AA ('ferries the load') to mRNA; also 'loads' the tRNA with the cognate AA. o Activating enzyme -- locks in energy for formation of peptide bond o AA-tRNA synthase (or synthetase) -- does accurate matching of AA and tRNA; ensures specificity o Charging enzyme -- both loads up AA and activates it -- locks in energy for formation of peptide bond. how RNA makes protein: need mRNA, tRNA, and ribosomes (containing rRNA) Where do tRNA and rRNA come from? All RNA's are encoded by DNA just like mRNA is. So there are genes for tRNA's and rRNA's on DNA tRNA and rRNA are not used as templates. When tRNA and rRNA genes are transcribed, the products fold up, associate with proteins if needed (for rRNA) and do their jobs. These RNA's are not translated -- they are agents that help in the translation of other RNA's (mRNA). different types of RNA have different half lives o tRNA and rRNA are relatively long-lived o prokaryotic mRNA is relatively short-lived, eukaryotic mRNAs vary

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What does it take to make one peptide? o mRNA -- You need one kind to make one polypeptide. o rRNA -- You need several kinds (3-4) to make one ribosome; exact # depends on whether it's a eukaryotic or prokaryotic ribosome. o tRNA -- You need one complete set (to pick up all amino acids and read all codons but stops). This is assuming the peptide you are making contains all 20 amino acids and the mRNA uses all possible codons. We calculated above that will you need about 30 different kinds. what if you want to make a different protein? o will need a different mRNA unless it's polycistronic o don't need a new set of rRNAs/a ribosome o don't need a new set of tRNAs Summary: mRNA is the software which is unique to the protein being made; tRNA & rRNA (& associated proteins) are the hardware that can be used to make any protein

Lecture 15 REREAD THIS ENTIRE DAMN LECTURE  Nonsense vs Mis-sense. A mutation that generates a stop codon is sometimes called a "nonsense" mutation; one that changes one amino acid to another is called a "mis-sense" mutation.  mutation level is low but nonzero  mutations are important b/c

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source of evolutionary diversity source of individual (and nonfunctional) diversity  Mutation leads to variations in all DNA, including noncoding DNA that does not affect the phenotype. This has little or no functional consequences, but the variations come in handy for tracing evolutionary lines of descent and making identifications Regulation of Enzyme Synthesis in Prokaryotes o glycolytic enzymes - always needed o beta-galactosidase  catalyzes first step in catabolic (breakdown) pathway  only needed if lactose present (when lactose needs to be broken down)  enzyme level should be low until lactose added to medium o TS (trp synthetase)  catalyzes last step in anabolic (synthetic) pathway  only needed if trp low or absent (when trp must be synthesized in order to make proteins  enzyme level should be high until trp added to medium 

How is co-ordinate control achieved? o Genes regulated together are linked -- genes to be controlled co-ordinately (turned on and off together) are next to each other on the DNA. o Polycistronic mRNA. The linked genes are transcribed as a unit to give one single mRNA . One mRNA is made per operon (not one mRNA per gene), because all the genes in a cluster share a single promoter. An mRNA able to code for several peptides (mRNA that comes from several genes) is called polycistronic mRNA. (cistron = another term for gene). o operon: group of linked structural (protein coding) genes that share common regulatory sites and that are transcribed as a single unit  each operon has at least two regulatory sites linked to the structural genes (that is, located close by on the same DNA)  promoter: binds RNA polymerase; determines how much mRNA can be made when operon is 'on'  operator: binds repressor protein; determines to what extent operon is 'on' or 'off' o punctuation - Numbers: Number of transcription starts (Promoters) for a message is one; number of translation starts will be greater than one on a polycistronic mRNA.

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Ps can be strong or weak Weak Promoter → little (or infrequent) RNA polymerase binding → low levels of transcription → low levels of corresponding protein.  Strong Promoter → lots of (or frequent) RNA polymerase binding → high levels of transcription → high levels of corresponding protein.  Why does strength of promoter matter? The strength of the promoter determines how much mRNA can be made (in an individual cell). Actual amount of mRNA made at any time (in total culture) depends on both strength of promoter and extent of repression or induction. o operator/repressor make an on/off switch  Repressor is a protein that binds to operator and prevents RNA polymerase from binding to DNA and transcribing the operon  There is a different repressor protein (& operator) for each operon. Repressor binds to specific sequence of DNA found in its respective operator.  The terms 'repressor' and 'repressor protein' are used interchangeably.  Synthesis of repressor protein is constitutive -- gene is always on  State (conformation) of repressor protein varies, not the amount  Repressor protein is allosteric (has two forms) -- one that sticks to the operator and blocks transcription (active form = rectangle) and one that doesn't (inactive form = circle).  Repressor binds effector (inducer or co-repressor). Each repressor/regulator protein is unique in that it binds the proper co-repressor or inducer (see below) as well as the proper operator.  Effector determines which form the repressor is in.  The amount of repressor protein present doesn't change (see above); the form repressor is in does change.  The small molecule effector (inducer or co-repressor) shifts the balance between the two forms (rectangle and circle) thus shifting the ...