Overview of Molecular Microbiology Selected Information from Chapters 4 8 10 Only responsible for Power Points Central Dogma of Molecular Biology Slide 1 Synthesis of Three Types Macromolecules C T A A G G T C A T C G T A G C Replication Transcription A A C G DNA G C C G G T A C A T A C T T G A G C T C A G T T A T Dark green strand is template for RNA synthesis C C G T G A G C T T DNA polymerase 5 T A 3 C A C T G C A C T G A T G G C G A C U U U C U C C T G A G C A A G G A G A G C C C T T C U Messenger RNA is template for protein synthesis tRNA mRNA A 5 G A C U G C G C G T C A C G G A C C A G C 3 Ribosome 2015 Pearson Education Inc 3 G C 5 Figure 4 3 RNA polymerase A Translation U G G A G G C C U G A U U G G U G A G G C G mRNA 5 G A C Protein T G DNA is template for own replication The anti sense strand of DNA is transcribed into a mRNA The mRNA nucleotide sequence is translated into the amino acid sequence of the polypeptide chain GENE the nucleotide sequence coding for a single polypeptide chain or a ribosomal RNA or a t RNA Contrast of information transfer in prokaryotes and eukaryotes Prokaryote A single mRNA often contains more than one coding region such mRNAs are called polycistronic Single Messenger RNA may carry several genes coding regions In Eukaryotes the DNA contains introns and exons The Primary RNA transcript must undergo processing which means the introns must be removed and the exons are spliced back together to make mature mRNA that is transported to the cytoplasm The mRNAs of eu are almost always monocistronic only code for one polypeptide chain Eukaryotic mRNA Spliceosome removes introns and joins adjacent exons to form mature RNA Two steps prior to splicing capping before transcription is complete Addition of a methylated guanine nucleotide a the 5 phosphate end promotes formation of initiation complex between mRNA and ribosome later Trimming 3 end and adding poly A tail not translated stabilizes mRNA Must be removed before mRNA is degraded Polymer of deoxyribonucleotides held together by 3 5 phosho diester bonds DNA is double stranded anti parallel with the two strands held together by hydrogen bonding between the bases The hydrogen bonds between the bases will break as temperature is raised but not the covalent bonds Tm value melting temperature This will separate the strands and DNA is said to melt Complementary and antiparallel nature of DNA Note that one chain ends in a 5 phosphate group whereas the other ends in a 3 hydroxyl The red bases represent the pyrimidines cytosine C and thymine T and the yellow bases represent the purines adenine A and guanine G DNA Double Helix Pyrimidine in red Purine in yellow Major and Minor groove S P backbone one in green and one in blue DNA in eukaryotes associates with basic proteins called histones to form nucleosome In Prokaryotes the DNA is also double stranded but the ends are closed to make a circle In both cases the DNA is highly coiled Nonchromosomal genetic elements include Viruses either DNA or RNA viroids small pieces of RNA Plasmids small genetic elements that exist and replicate separately from the major bacterial chromosome Most are circular polynucleotides that do not usually cause cell damage and they do not have extra cellular forms Some can integrate into host DNA DNA replication is semiconservative If DNA is labeled with heavy N15 Nitrogen and then the cells are allowed to replicate 1st generation in light nitrogen N14 the DNA in the daughter cells will be of a hybrid density Messelson and Stahl experiment Meselson Stahl DNA Replication is Semiconservative Cells grown in 15N All DNA heavy Cells grown in 14N 1st gen hybrid Cells continue in 14N 2nd gen hybrid light DNA polymerase can not replicate a strand de novo because it requires a primer Therefore a small RNA primer is synthesized by primase in the 5 to 3 direction initiates DNA synthesis by DNA polymerase III DNA polymerase III can only synthesize in the 5 to 3 direction so it can do so continuously on the leading strand but must synthesize DNA in segments in the lagging strand These segments are called Okazaki fragments each must be separately primed but in contrast the leading strand is only primed once DNA polymerase III is synthesizing DNA in the 5 3 direction toward the RNA primer of a previously synthesized fragment on the lagging strand On reaching the fragment DNA polymerase I replaces III removes the primer of the previous fragment as it synthesizes DNA DNA ligase then replaces DNA polymerase I seals the gap by linking the 3 OH to the 5 phosphate DNA Unwinding Helicase dnaB unwinds the DNA double helix Energy provided by ATP Move along helix and separate strands Single stranded regions complexed with ssb proteins which stabilize single stranded form DNA Replication Lagging strand DNA polymerase III is synthesizing DNA in 5 3 direction toward RNA primer of previously synthesized fragment On reaching fragment DNA pol III leaves is replaced by DNA pol I DNA pol I removes RNA primer exonuclease while continuing to fill in nucleotides 5 3 DNA ligase seals the gap Complexity of Process Molec Bio I Replisome 2 copies of pol III plus helicase primase primosome and many copies of ssb Tau subunits hold 2 DNA polymerase assemblies and helicase together Upstream of replisome DNA gyrase removes supercoils 2 pol III replicate 2 individual strands in opposite directions thus laggging strand loops around so replisome moves in same direction along DNA RNA polymerase consists of a sigma subunit core enzyme 2 For RNA synthesis to begin the RNA polymerase must recognize the proper region to start This region is called the promoter it is recognized by the sigma factor of RNA Polymerase Consensus sequence similar in many different species 35 and 10 Pribnow Box Many mRNAs made off of DNA need to know where to start transcription where to stop Transcription Initiation by Sigma Recognition Binding to Promoter Transcription Core enzyme sigma bind Sigma recognizes promoter consensus Transcription begins After a few nucleotides sigma is released Chain termination site Polymerase RNA released Chain Termination Inverted repeats will hydrogen bond together in transcribed DNA form a stem loop structure in RNA that terminates transcription Stem loop in RNA is upstream from a run of uracils Other mechanisms covered in MBII Rho dependent Anticodon Codon Recognition Steps in Translation Initiation Elongation Termination Mutations involving one base pair are called point