A-T C-G1. RNA differs from DNA a.

The polynucleotide structure of RNA is similar to DNA except that RNA contains the sugar ribose rather than deoxyribose and uracil !" rather than thymine. A small amount of thymine is present in tRNA." Semiconservative replication, although the parental duplex is separated into t#o hal$es and% therefore% is not &conser$ed' as an entity"% each of the indi$idual parental strands remains intact in one of the t#o ne# duplexes (nitiation of DNA replication re)uires the recognition of the origin of replication by a group of proteins that form the prepriming complex. DNA helicases bind to ssDNA near the replication for*% and then mo$e into the neighboring doublestranded region% forcing the strands apart+in effect% un#inding the double helix. ,ingle-stranded DNA-binding ,,-" proteins bind to the ssDNA generated by helicases. These proteins not only *eep the t#o strands of DNA separated in the area of the replication origin% thus pro$iding the single-stranded template re)uired by polymerases% but also protect the DNA from nucleasesthat degrade ssDNA. leading strand. in the direction of the ad$ancing replication for* lagging strand. a#ay from the replication for* The RNA% then% is complementary to the DNA template antisense" strand and identical to the coding sense" strand% #ith ! replacing T. RNA primer+that is% a short% double-stranded region consisting of RNA base-paired to the DNA template% #ith a free hydroxyl group on the /0-end of the RNA strand. This hydroxyl group ser$es as the first acceptor of a deoxynucleotide by action of DNA polymerase. Primase.A specific RNA polymerase% called primase DnaG"%synthesi1es the short stretches of RNA approximately ten nucleotides long" that are complementary and antiparallel to the DNA template The primosome ma*es the RNA primer re)uired for leading strand synthesis% and initiates 2*a1a*i fragment formation in lagging strand synthesis. DNA polymerase III:DNA chain elongation is cataly1ed by DNA polymerase (((. !sing the /0hydroxyl group of the RNA primer as the acceptor of the first deoxyribonucleotide% DNA polymerase ((( begins to add nucleotides along the single-stranded template that specifies the se)uence of bases in the ne#ly synthesi1ed chain. DNA polymerase (((is a highly &processi$e' en1yme+that is% it remains bound to the template strand as it mo$es along% and does not diffuse a#ay and then rebind before adding each ne# nucleotide. The processi$ity of DNA polymerase ((( is the result ofits 3subunit forming a ring that encircles and mo$es along the template strand of the DNA% thus ser$ing as a sliding DNA clamp. The ne# strand gro#s in the 405/0 direction% antiparallel to the parental strand. The nucleotide substrates are 40-deoxy ribo nucleoside triphosphates. 6yrophosphate 66i" is released #hen each ne# deoxynucleoside monophosphate is added to the gro#ing chain see 7igure 89.14". :ydrolysis of 66i to 86i means that a total of t#o high-energy bonds are used to dri$e the addition of each deoxynucleotide

To ensure replication fidelity% DNA polymerase (((has% in addition to its 405/0 polymeraseacti$ity% a &proofreading' acti$ity As each nucleotide is added to the chain% DNA polymerase ((( chec*s to ma*e certain the added nucleotide is% in fact% correctly matched to its complementary base on the template. (f it is not% the /0540 exonucleaseacti$ity corrects the mista*e DNA polymerase (((continues to synthesi1e DNA on the lagging strand until it is bloc*ed by proximity to an RNA primer. ;hen this occurs% the RNA is excised and the gap filled by DNA polymerase ( DNA ligase The final phosphodiester lin*age bet#een the 40-phosphate group on the DNA chain synthesi1ed by DNA polymerase (((and the /0-hydroxyl group on the chain made by DNA polymerase (is cataly1ed by DNA ligase 7igure 89.8<". The =oining of these t#o stretches of DNA re)uires energy% #hich in most organisms is pro$ided by the clea$age of AT6 to A>6 ? 66i 6ol @ epsilon"AThought to elongate leading strand eu*aryote" The RNAprimers are remo$ed by nucleases e.g.% RNase :"% and then the resulting gaps are filled#ith the appropriate deoxyribonucleotides by another DNA polymerase. 7inally% the2*a1a*i fragments are =oined by DNA ligase%an en1yme that cataly1es formation of phosphodiester bonds bet#een t#o polynucleotide chains Gene contain a promotor region #ith #hich th eRNA polymerase bind. ;hen RNA polymerase binds to apromoter%local un#inding of the DNA helix occurs% so that the DNA strands partially separate. The polymerase then begins transcription% copying the template strand Enhancers are DNA se)uences that function in the stimulationof the transcription rate. (t begins #ith a start codon A!G" near the 4 end of the mRNA. (t ends #ith a termination stop" codon !GA% !AG%or!AA" near the / end. Initiation of translation (n eu*aryotes% methionyl-tRNAimet binds to the small ribosomal subunit. a. The 40 cap of the mRNA binds to the small subunit% and the first A!G codon base pairs #ith the anticodon on the methionyl-tRNAimet b. The methionine that initiates protein synthesis is subse)uently remo$ed from the N terminus of the polypeptide d. 6ro*aryotes do not contain a 40cap on their mRNA. An mRNA se)uence upstream from the translation start site the ,hine-Dalgarno se)uence" binds to the /0 end of 1B, ribosomal RNA rRNA" to position the small ribosomal subunit on the mRNA. 8. The large ribosomal subunit binds% completing the initiation complex. a. The methionyl-tRNAi>et is bound at the 6 peptidyl" site of the complex. b. The A acceptor or aminoacyl" site of the complex is unoccupied. /. (nitiation factors (7s"% AT6% and guanosine triphosphate GT6"are re)uired for formation of the initiation complex. a. Theinitiation factors are designated (7-1% (7-8% and (7-/ in pro*aryotes. (n eu*aryotes% they are designated e(7-1% e(7-8% and so on. ,e$en or more may be present.

b. Release of the initiation factors in$ol$es hydrolysis of GT6 to guanosine diphosphate GD6" and 6i Elongation of polypeptide chains 1. The addition of each amino acid to the gro#ing polypeptide chain in$ol$es binding of an aminoacyl-tRNA at the A site% formation of a peptide bond% and translocation of the peptidyltRNA to the 6 site. 8. Binding of aminoacyl-tRNA to the A site a. The mRNA codon at the A site determines #hich aminoacyl-tRNA #ill bind. 1" The codon and the anticodon bind by base pairing that is antiparallel. 8" (nternal methionine residues in the polypeptide chain are added in response to A!G codons. They are carried by tRNAm>et% a second tRNA specific for methionine. b. An elongation factor C7" C7-Tu in pro*aryotes and C7-1 in eu*aryotes" and hydrolysis of GT6 are re)uired for binding. /. ormation of a peptide !ond a. A peptide bond forms bet#een the amino group of the aminoacyl-tRNA at the A site and the carbonyl of the aminoacyl group attached to the tRNA at the P site. 7ormation of the peptide bond is cataly1ed by peptidyl transferase% #hich is rRNA. b. The tRNA at the 6 site no# does not contain an amino acid. (t is DDuncharged.EE c. The gro#ing polypeptide chain is attached to the tRNA in the A site. F. "ranslocation of peptidyl-tRNA a. The peptidyl-tRNA along #ith the attached mRNA" mo$es from the A site to the 6 site% and the uncharged tRNA is released from the ribosome. An elongation factor C7-8 in eu*aryotes or C7-G in pro*aryotes" and the hydrolysis of GT6are re)uired for translocation. b. The next codon in the mRNA is no# in the A site. c. The elongation and translocation steps are repeated until a termination codon mo$es into the A site. 7. "ermination of translation 1. ;hen a termination codon #$A, #A$, or #AA" occupies the A site% release factors cause the ne#ly synthesi1ed polypeptide to be released from the ribosome. 8. The ribosomal subunits dissociate from the mRNA. Point m%tations occur #hen 1 base in DNA is replaced by another% altering the codon in mRNA a. Silent m%tations do not affect the amino acid se)uence of a protein e.g.% CGA to CGG causes no change because both codons specify arginine". b. &issense m%tations result in one amino acid being replaced by another e.g.% CGA to CCA causes arginine to be replaced by proline". c. Nonsense m%tations result in premature termination of the gro#ing polypeptide chain e.g.% CGA to !GA causes arginine to be replaced by a stop codon". F. Insertions occur #hen a base or a number of bases are added to DNA. They can result in a protein #ith more or fe#er amino acids than normal. 4. Deletions occur #hen a base or a number of bases are remo$ed from DNA. They can result in a protein #ith fe#er or more amino acids than normal. B. rameshift m%tations occur #hen the number of bases added or deleted is not a multiple of three. The reading frame is shifted so that completely different sets of codons are read beyond the point at #hich the mutation starts