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Lecture 13

BLG 151 Lecture 13: Chapter 13 part-2 notes

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Ryerson University
BLG 151
Martina Hausner

Microbiology Chapter 13: Part 2 13.3 DNA Replication in Bacteria Flow of Genetic Information: Replication/Transcription and Translation  Replication: synthesis of DNA from DNA (parent)  Transcription: synthesis of RNA from DNA  Translation: synthesis of protein from mRNA DNA Replication  Complex process involving numerous proteins which help ensure accuracy  The 2 strands separate, each serving as a template for synthesis of a complementary strand  The synthesis is semi-conservative; each daughter cell obtains one old and one new strand DNA Replication is Semi-Conservative  Each of the 2 progeny DNA molecules consists of one new strand and one old strand  Very accurate – the error rate is about 1 in 10 or 100  Very rapid – 750 to 1000 base pairs per second in bacteria. And 100 base pairs per second in eucaryotes Bacterial DNA Replication Initiates from a Single Origin of Replication  DNA in most bacteria is circular  Bidirectional replication from a single origin  Replication fork is where DNA is unwound o Replicon: the portion of the genome with the origin, replicated as a unit (entire genome in bacteria) The Replication Machinery  Consists of at least 30 proteins in e.g. E.coli, many are different from archaeal and eukaryotic replication enzymes Replication Machinery – Polymerases  DNA polymerase catalyzes synthesis of complementary strand of DNA  DNA synthesis in 5’ to 3’ direction forming phosphodiester bonds  Major enzyme?  DNA polymerase (5 polymerases; polymerase III, assested by Pol I, plays a major role in replication)  Polymerases require:  A template ( read in 3’ to 5’ direction) – directs synthesis of complementary strand  A primer – DNA or RNA strand to provide a free 3’-hydroxul group to which nucleotides can be added  Deoxynucleotide triphosphates – dNTPs (dATP, DTTP, DCTP, dGTP) o Deoxynucleotide triphosphates are linked by phosphodiester bonds between the hydroxyl group at the 3’ end of the growing strand and the phosphate closest to the 5’ C of the incoming deoxynucleotide (alpha phosphate) Replication Machinery – The Polymerase Pieces  DNA polymerase holoenzyme is a complex of 10 proteins  3 proteins form core enzyme  catalyze DNA synthesis, proofread for fidelity  Polymerase III (Pol III) holoenzyme contains  Beta clamp which tethers the enzyme to DNA  A complex of proteins called the T (tau) complex clamp loader o This includes proteins responsible for loading the beta clamp onto DNA o The tau proteins in the complex hold the holoenzyme together.  Both strands of DNA are bound by 1 Pol III  Replisome – complex of proteins involved in replication (includes Pol III) Other Replisome Proteins  SSBs – single stranded binding proteins: keep strands apart for replication to occur  Helicase: unwinds DNA strands, use ATP  Primase: (RNA polymerase, can initiate RNA synthesis without an existing 3’-OH) part of primosome, synthesizes short complementary strands of RNA primers (~10 nucleotides) for DNA polymerase  Topoisomerase: (gyrase) breaks one strand of DNA; relieves tension from rapid unwinding of double helix, prevents supercoiling  DNA gyrase: also introduces negative supercoiling, helps compact bacterial chromosome  Leading strand: synthesized at its 3’ end as DNA unwinds  Lagging strand: no 3’-OH, synthesized discontinuously in the 5’ to 3’ direction, opposite to the movement of the replication fork  Okazaki fragments; a new RNA primer is needed for synthesis of each Okazaki fragment Events at the Replication Fork in E. coli  DNA polymerase synthesis is in 5’ to 3’ direction only  Bacterial initiator protein DnaA binds oriC locus (origin or replication, AT rich) causing bending and separation of strands  DnaB helicase and topoisomerases separate strands, SSB attach, keep strands separated  Primase synthesizes RNA primer  Lagging and leading strand is synthesized by Pol III  On leading strand, replication is continuous  The ladding strand is synthesized in short fragments called Okazaki fragments -A new primer is needed for the synthesis of each Okazaki fragment  Pol I removes RNA primers and Ligase joins DNA fragments  DNA polymerase I removes RNA primers by removing nucleotides starting at the 5’end moving towards the 3’ end  5’ to 3’ exonuclease activity, fills gaps between Okazaki fragments with 3’-OH from deoxynucleotide  Okazaki fragments are joined by DNA ligase  phosphodiester bond between 3’- OH of growing strand and 5’-phosphate of Okazaki fragment Linking the Fragments  DNA ligase forms a phosphodiester bond between 3’-hydroxyl of growing strand and 5’- phosphate of an Okazaki fragment Proofreading  Carried out by DNA polymerase III  Removal of mismatched base from 3’end of growing strand by 3’ to 5’ exonuclease activity of Pol III enzyme  This activity is not 100% efficient Termination of Replication in E. coli  Replication stops when replisome reaches termination site (ter) on DNA; protein (tus) binds to ter and stops progression of forks  In many bacteria replication stops spontaneously when the forks meet  Catenanes (interlocked chromosomes) form when the two circular daughter chromosomes do not separate  Topoisomerases temporarily break the DNA molecules so the strands can separate  Dimerized chromosomes (2 chromosomes joined together to form a single chromosome 2x long) result from DNA recombination  Recombinase enzymes (e.g. XerCD in E.coli) catalyze a cross over that separates the two chromosomes 13.4 Bacterial Gene Structure Gene  Polynucleotide sequence that codes for a functional product (rRNA, tRNA or mRNA – protein)  Transcription yields three major type of RNA molecules  Messenger RNA: mRNA molecules arise from transcription of protein-coding genes. They are translated into protein with the aid of the other two major types of RNA  Transfer RNA: tRNA molecules carry amino acids to the ribosome during translation;  Ribosomal RNA: rRNA molecules have several functions, including catalyzing peptise bond formation mRNA Genes – Protein Coding Genes  Promoter: binding site for RNA Pol; neither transcribed nor translated  Leader: at the transcription start site (+1), transcribed into mRNA, not translated. In bacteria, it includes the Shine-Dalgarno sequence (needed for initiation of translation)  Coding region: begins with 3’-TAC-5’  start codon 5’-AUG-3’, sequence of amino acids for the rest of the protein. Ends with a stop codon – signals the end of the protein.  Template strand: directs mRNA synthesis, beginning of gene at the 3’ end  Sense (coding) strand: complementary to template strand, same nucleotide sequence as mRNA (except in DNA bases)  Trailer: transcribed, but not translated. Contains a sequence which prepares the RNA Pol for release from template strand.  Terminator: signals the DNA polymerase to stop transcription tRNA and rRNA Genes  DNA sequences that code for tRNA and rRNA are considered genes  Several tRNA or rRNA genes are often transcribed together under the control of a single promoter  Genes coding for tRNA may code for more than a single tRNA molecule or type of tRNA  Genes coding for rRNA are transcribed as signle, large precursor and cut to yield rRNAs (and sometimes tRNAs)  Spacers between the coding regions are removed after transcription, some by the use of special ribonucleases called ribozymes 13.5 Transcription in Bacteria Flow of Genetic Information: Replication/Transcription and Translation  Replication: synthesis of DNA from DNA  Transcription: synthesis of RNA from DNA  Translation: synthesis of protein from mRNA Transcription in Bacteria  Synthesis of RNA under the direction of DNA  The RNA product has a sequence complementary to DNA template strand  Generates three major kinds of RNA:  Transfer RNA tRNA – carries amino acids during protein synthesis  Ribosomal RNA rRNA – are components of ribosomes  Messenger RNA mRNA – bears the message for protein synthesis  Involves 3 stages:  Initiation  Elongation  Termination  Bacterial genes encoding proteins involved in a related process are often located close to each other and are transcribed from a single promoter (transcriptional unit: operon)  Transcription of an operon yields an mRNA consisting of a leader followed by one coding region, which is separated from the second coding region by a spacer, and so on, with the final sequence of nucleotides being the trailer. Such mRNAs are said to be polycistronic mRNAs (bacteria, archaea) RNA is Synthesized by a Single RNA Polymerase in Bacteria  Bacterial RNA polymerase – 5 polypeptide chains of  RNA polymerase core enzyme – 2x  RNA holoenzyme contains core enzyme plus (sigma) factor – recognizes promoter sequence and initiates transcription 70  factor recognizes the most common promoter sequence Sigma 70 Promoter – Most Common One  Where does transcription start? At the promoter. Many bacterial promoters are recognized by the sigma factor which positions RNA Pol at the
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