BIO 326R 1st Edition Lecture 10 Outline of Last Lecture I. HistoryII. Bacterial chromosomes Replicationa. Steps to replicationIII. Initiation of synthesis and synthesis mechanismOutline of Current Lecture I. Why does replication not continually initiatea. Hemimethylation and dnaAb. Cell sizeII. Proofreadinga. Base pairingb. Exonuclease activityc. Post synthesis mismatch repairIII. Terminating ReplicationIV. Transcriptiona. Stepsb. Types of RNAc. RNAPV. Initiationa. Promoter sequenceCurrent LectureWhy not continuous initiation of replication?- DnaA does not bind to hemimethylated DNAo Old strand is methylated, new is unmethylatedo SeqA= binds hemimethylated oriC prevents DnaA binding- Cell sizeo Concentration of DnaA ([DnaA]) depends on the size of the cell Large cell= growth and low [DnaA]no need to initiate replicationProofreading- 1/10^9 to 1/10^10 errors introduced—how so low?o Base pairing is specific and preciseo DNA polymerase I and III have 3’5’ exonuclease activity Proofreading—exonuclease cleaves incorrect nucleotideThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute. 3’5’ because as it is synthesized 5’3’ exonuclease “backs up” to cleave the nucleotideo Post synthesis mismatch repair system Recognizes mismatches on nonmethylated (new) strand and cleaves 1000 times more mutagenic—1/10^6 – 1/10^7- Bacteria wants to be more mutagenic because this normally allows more resistance to antibioticsTerminating Replication- Replicating strands meet about 180 degrees from OriC- Stop at specific ter (termination) sequences- Final step is a double strand breako Cleave double strand, pass through the other chromosome, relegate like you have two rings liked together—cut both to “unhook” and then rejoin the cut portionTranscription- transcription= RNA from DNA- synthesized RNA is called the transcripto 5’3’ – coding strand (homologous sequence to synthesized RNA)3’5’ – template strand (strand that is copied to make RNA)- RNA differs from DNA in that:o Uracil instead of thymineo Single strand instead of double strando Ribose instead of deoxyribose Sugar moiety differs—2’ hydroxyl present and less stable- 3 steps to make RNA (will discuss each in detail later)o Initiationo Elongationo Termination- Location on DNA where RNA starts is called +1- 4 types of RNA (slides have more information on each)o Messenger RNA (mRNA) Encodes proteino Ribosomal RNA (rRNA) Involved in translation Most prevalent (80%)o Transfer RNA (tRNA) Carries amino acids for translationo Small regulatory RNA (sRNA) Regulate transcription and translation- RNA Polymerase (RNAP)o Enzyme that makes RNAo Common antibiotic targeto About 2,000 molecules per cello 2 forms—holoenzyme= whole enzyme Core enzyme- Alpha- 2 subunits, recognize promoter element- Beta- regulates polymerase activity- Beta’- binds DNA- W- restores RNAP to active conformation Sigma factor/subunit- Directs RNAP to specific promoter sequences- Multiple sigma factors (5-30)o Each have a preference for a different sequenceInitiation- How does RNAP know where to start?o Promoter sequence= specific sequence that precedes (upstream) a transcript Sequence is not absolute, but there is a general pattern of sequence Consensus promoter sequence in E. coli is recognized by sigma factor 70 (housekeeping promoter)- Recognizes promoter sequence- Sequence governs promoter
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