Chapter 26: RNA Metabolism Flashcards

Question

polynucleotide phosphorylase

Answer 1

* bacterial enzyme * first nucleic acid–synthesizing enzyme discovered * DNA polymerase followed soon thereafter * it is not template-dependent * uses the 5'-diphosphates of ribonucleosides as substrates * can't act on the homologous 5'-triphosphates or on deoxyribonucleoside 5'-diphosphates * RNA polymer formed contains the usual 3',5'-phosphodiester linkages, which can be hydrolyzed by ribonuclease * readily reversible. can be pushed in the direction of breakdown of the polyribonucleotide by increasing the phosphate concentration increasing the phosphate concentration * probable function is the degradation of mRNAs to nucleoside diphosphates * polymer it forms does not have a specific base sequence * base composition of the resulting polymer reflects nothing more than the relative concentrations of the 5'-diphosphate substrates in the medium * used in the laboratory to prepare RNA polymers

Answer 2

* RNA-dependent DNA polymerase * contains contain Zn²⁺ * RNA viruses are the source of most RNA-dependent polymerases characterized so far * RNA viruses that contain reverse transcriptases are known as retroviruses * enzyme is most active with the RNA of its own virus, but can be used experimentally to make DNA complementary to a variety of RNAs (cDNA) * structure * has an α and β subunit * α is simply a proteolytic fragment of the β subunit * enzyme catalyzes three different reactions, in separate active sites on the protein * RNA-dependent DNA synthesis * RNA degradation * DNA-dependent DNA synthesis **Retroviral Infection Pathway** * On infection, the singlestranded RNA viral genome and the enzyme enter the host cell * the genome has a tRNA (picked up from a former host cell) already base-paired to the viral RNA * facilitates immediate conversion of viral RNA to double-stranded DNA * reverse transcriptase synthesizes DNA strand complementary to viral RNA * degrades the RNA strand of the viral RNA-DNA hybrid * replaces degraded RNA strand with complementary DNA * new DNA strand is synthesized in the 5'→3' direction * doesn't have 3'→5' proofreading exonucleases * has a high error rate (about 1 per 20,000) * seems to be a feature of most enzymes that replicate the genomes of RNA viruses * higher mutation rate yields faster rate of viral evolution * DNA enters the nucleus and is integrated into the host genome * At each end of the linear RNA genome are long terminal repeat (LTR) sequences of a few hundred nucleotides * they facilitate integration of the viral chromosome into the host DNA and contain promoters for viral gene expression * integration is catalyzed by a virally encoded integrase * integration is similar to the insertion of transposons in bacterial chromosomes * a few base pairs of host DNA are duplicated at integration site, forming short repeats of 4-6 bp at each end of the inserted retroviral DNA * During transcription * Retroviruses typically have three genes and a φ sequence * gag encodes the interior core of the viral particle * pol encodes * protease that cleaves the long polypeptide * integrase that inserts the viral DNA into the host chromosomes * reverse transcriptase * env encodes the proteins of the viral envelope * φ packages retroviral RNAs into mature viral particles * Transcription produces a primary transcript encompassing the gag, pol, and env genes * Splicing of the primary transcript yields an mRNA derived from the env gene * translation * Translation produces a polyprotein, derived from the gag and pol genes, which is cleaved into six distinct proteins * Env primary script is also translated into a polyprotein, then cleaved to generate viral envelope proteins * new viruses are formed and released by cell lysis (right)

Answer 3

* Some well-characterized eukaryotic DNA transposons have a structure very similar to that of retroviruses **Retrotransposons** * encode an enzyme homologous to the retroviral reverse transcriptase * coding regions are flanked by LTR sequences * transpose from one position to another in the cellular genome * have an RNA intermediate * uses reverse transcriptase to make a DNA copy of the RNA * followed by integration of the DNA at a new site * the copy distinguishes them from bacterial transposons, which move as DNA directly from one chromosomal location to another * followed by integration of the DNA at a new site * lack an env gene * can be thought of as defective viruses, trapped in cells * suggest that reverse transcriptase is an ancient enzyme that predates the evolution of multicellular organisms **Introns** * group I and group II introns are also mobile genetic elements * they encode DNA endonucleases that promote movement * group I * process is called homing * During genetic exchanges, endonucleases promote insertion of the intron into an identical site in another DNA copy of a homologous gene that does not contain the intron * group II * process called retrohoming * occurs through an RNA intermediate * endonucleases have associated reverse transcriptase activity * proteins can form complexes with the intron RNAs after introns are spliced from the primary transcripts * the homing process involves insertion of the RNA intron into DNA and reverse transcription of the intron * rarely the intron may insert itself into a new location in an unrelated gene * can kill the host cell * or lead to the evolution and distribution of an intron in a new location * the introns structures and mechanisms support the idea that at least some originated as molecular parasites whose evolutionary past can be traced to retroviruses and transposons * PDF pg. 1121, figure 26-37

Answer 4

**Telomeres** * structures at the ends of linear eukaryotic chromosomes * consist of many tandem copies of a short oligonucleotide sequence * sequence usually has the form T_xG_y in one strand and C_yA_x in the complementary strand * x and y are typically in the range of 1 to 4 * vary in length * The TG strand is longer than its complement, leaving a region of single-stranded DNA of up to a few hundred nucleotides at the 3' end * ends of a linear chromosome are not readily replicated by cellular DNA polymerases * beyond the end of a linear DNA molecule no template is available * telomerase solves this problem by adding telomeres to chromosome ends **telomerase** * contains both RNA and protein components * RNA component is about 150 nucleotides long and contains about 1.5 copies of the appropriate C_yA_x telomere repeat * acts as a template for synthesis of the T_xG_y strand * Telomerase acts as a cellular reverse transcriptase that provides the active site for RNA-dependent DNA synthesis * copies only a small segment of RNA that it carries within itself * requires the 3' end of a chromosome as primer * proceeds in the usual 5'→3' direction * after extension of T_xG_y strand the complementary C_yA_xstrand is synthesized by cellular DNA polymerases, starting with an RNA primer * in lower eukaryotes * single-stranded region is protected by specific binding proteins in many * in higer eukaryotes * the single-stranded end is sequestered in a specialized structure called a T loop * folded back and paired with its complement in the double-stranded portion of the telomere * formation of T loop involves invasion of the 3' end of the telomere’s single strand into the duplex DNA * similar to the initiation of homologous genetic recombination * In mammals, the looped DNA is bound by two proteins, TRF1 and TRF2 * T loops protect the 3' ends of chromosomes, from nucleases and the enzymes that repair double-strand breaks

Answer 5

* replicate ssRNA chromosomes of viruses, which also function as mRNAs for the synthesis of viral proteins * All RNA viruses (except retroviruses) must encode thier own RNA-dependent RNA polymerase, because the host cells do not possess this enzyme * catalyzes the formation of an RNA complementary to the viral RNA, similar to DNA-dependent RNA polymerases * synthesis proceeds in the 5'→3' direction * lacks a separate proofreading endonuclease activity and has an error rate similar to that of RNA polymerase * specific for the RNA of their own virus * RNAs of the host cell are generally not replicated. * structure * has a molecular weight of 210,000 * consists of four subunits * One subunit (Mr 65,000) is the product of the replicase gene encoded by the viral RNA and has the active site for replication * other three subunits are host proteins normally involved in host-cell protein synthesis: * the E. coli elongation factors Tu (Mr 45,000) and Ts (Mr 34,000) (which ferry amino acyl–tRNAs to the ribosomes) * protein S1 (an integral part of the 30S ribosomal subunit). * These three host proteins may help the RNA replicase locate and bind to the 3' ends of the viral RNAs.

Answer 6

The RNA world hypothesis is now supported by at least five lines of evidence. 1. RNA catalysts exist. RNA thus clearly has the capacity to both store information and catalyze reactions. 2. the wide range of reactions for which RNA catalysts have been developed is sufficient to form the basis of a primordial metabolic system. 3. there are numerous extant RNAs that are likely vestiges of an RNA world, including ribozymes, retroviruses, RNA viruses, and retrotransposons. 4. an RNA catalyst is responsible for the synthesis of proteins 5. Finally, RNA catalysts with a capacity for self-replication are coming to light in the laboratory PDF PG 1124

Answer 7

* The four code letters of DNA (A, T, G, and C) in groups of three yield 4³ = 64 different combinations. * any given single-stranded DNA or mRNA sequence has three possible reading frames * Each reading frame gives a different sequence of codons * only one is likely to encode a given protein * codons are written in the 5'→3' direction * The third base of each codon plays a lesser role in specifying an amino acid than the first two * All the amino acids except methionine and tryptophan have more than one codon * initiation codon * AUG * most common signal for the beginning of a polypeptide in all cells * also codes Met residues in internal positions of polypeptides * termination/stop/nonsense codons * UAA, UAG, and UGA * signal the end of polypeptide synthesis * do not code for any known amino acid * open reading frame (ORF) * a reading frame without a termination codon among 50 or more codons * Long open reading frames usually correspond to genes that encode proteins * code is described as degenerate * amino acid may be specified by more than one codon * each codon specifies only one amino acid * genetic code is defined by two elements * the anticodons on tRNAs * tRNAs determine where an amino acid is placed in a growing polypeptide * the specificity of aminoacyl-tRNA synthetases * charges the tRNAs * determines the identity of the amino acid attached to a given tRNA * Codon bias * several codons encode the same amino acid and utilize multiple tRNAs * not all of the codons are used with equal frequency * The tRNAs for the frequently used codons are often present at much higher concentrations than other tRNAs **Changes in the Code** * To alter the code, changes must occur in the gene(s) encoding one or more tRNAs, altering the anticodon * this change leads to the insertion of an amino acid at a codon that does not specify that amino acid * most other changes occur in mitochondrial DNA * most involve termination codons * sometimes the effects are minor because the genes have multiple (redundant) termination codons

Answer 8

* codon: 5'→3' * anticodon: 3'→5' * by convention they are written in text beginning with 5'→3' * anticodon (5')ICG = 3'→GCI→5' * codon (5')CGA = 5'→CGA→3' * When several different codons specify one amino acid, the difference between them usually lies at the third base, at the 3' end * codons for most amino acids can be symbolized by XY^A_G or or XY^U_C * PDF PG 1141, table 27-4 * X and Y denote bases complementary to and capable of strong Watson-Crick base pairing with X' and Y' * Wobble bases—in the 3' position of codons and 5' position of anticodons—are shaded in white * The first base of the codon in mRNA (5'→3') pairs w/ 3rd base of the anticodon (5' end) * anticodons in some tRNAs include the nucleotide inosinate (I) * contains the uncommon base hypoxanthine * I can form hydrogen bonds with U, C, and A * pairings are much weaker than the regular ones * wobble/third base pairs loosely with the anticodon * permits rapid dissociation of the tRNA from its codon * If all three codon bases are in strong Watson-Crick pairing with the three anticodon bases, tRNAs would dissociate too slowly limiting rate of protein synthesis * Codon-anticodon interactions balance the requirements for accuracy and speed **Wobble Hypothesis** * first two bases of a codon form strong Watson-Crick base pairs with the anticodon and confer most of the coding specificity * first base of the anticodon * pairs with the third base of codon * by convention they are written in text beginning with 5'→3' * anticodon (5')**I**CG = 3'→GCI→5' * codon (5')CG**A** = 5'→CGA→3' * determines the # of tRNA that can match the codon * C or A * C binds with G * A binds with U * U or G * U bind with A or G * G bind with C or U * I * binds with A, U or C * When an amino acid is specified by several different codons, the codons that differ in either of the first two bases require different tRNAs * A minimum of 32 tRNAs are required to translate all 61 codons * 31 to encode the amino acids and 1 for initiation

Answer 9

* missense mutations * a single new base pair replaces another * In the third/wobble, position of the codon, single base substitutions produce a change in the encoded amino acid only about 25% of the time * changes are silent mutations, where the nucleotide is different but the encoded amino acid remains the same * transition mutation * most frequent missense mutation * a purine is replaced by a purine or a pyrimidine by a pyrimidine * A mutation in the first position of the codon will usually result in an amino acid coding change, but the change often involves an amino acid with similar chemical properties

Answer 10

* other two possible reading frames usually contain no useful genetic information * but a few genes are structured so that ribosomes “hiccup” at a certain point in the translation of their mRNAs, changing the reading frame from that point on * This appears to be a mechanism either to allow two or more related but distinct proteins to be produced from a single transcript or to regulate the synthesis of a protein * PDF PG 1142, FIGURE 27–9

Answer 11

* Some mRNAs are edited before translation * can involve the addition, deletion, or alteration of nucleotides in the RNA * Addition or deletion of nucleotides is most common in mitochondrial and chloroplast genomes * requires a gRNA, guide RNA * special class of RNA molecules encoded by these same organelles * sequences complementary to the edited mRNAs * gRNAs act as template for editing process * base pairing between the initial transcript and the guide RNA involves a number of G═U base pairs, common in RNA molecules * RNA editing by alteration of nucleotides involves the deamination of adenosine or cytidine residues, forming inosine or uridine * Inosine is interpreted as a G residue during translation * adenosine deamination reactions are carried out by adenosine deaminases that act on RNA (ADARs). * cytidine deaminations are carried out by the apoB mRNA editing catalytic peptide (APOBEC) family of enzymes, which includes the related activation-induced deaminase (AID) enzymes * Both groups of deaminase enzymes have a homologous zinc-coordinating catalytic domain