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RNA RNA is much like DNA except: •Nucleotides are not deoxy (that is they have a 2’ OH). •UTP is used instead of dTTP •Bases in RNA (especially tRNAs) are often modified (SLIDE) •RNA is usually single stranded •RNA can be edited by removal or addition of bases Content of RNA in a typical E. coli cell: Half lives: mRNA(minutes), tRNA(hrs/days), rRNA(hrs/days) •In prokaryotes there is one RNA polymerase (RNAP ) that is responsible for the synthesis of mRNA, tRNA and rRNA. •Eukaryotes have 3 RNAPs: I: synthesizes rRNA II: synthesizes mRNA III: synthesizes tRNA Other differences between prokaryotic and eukaryotic transcription: Prokaryotic Eukaryotic Place cytoplasm nucleus Structure polycistronic or monocistronic monocistronic Translation coupled separate from transcription Transcription There is one RNA polymerase in bacteria responsible for mRNA, tRNA and rRNA synthesis. It is not the same as the one that is involved in priming DNA synthesis. Promoter structure •Two parts -35 region and the -10 region -For housekeeping genes the promoter is similar to: TTGACA-N14-TATAAT •These regions are -35 bp and -10 bp to the left of the start of the mRNA which begins at a site called the +1 site. Determination of important binding sites by finding consensus sequences First consensus slide RNAP composition: 2 x subunit (gene: xx)--These bind regulatory sequences near promoters 2 x subunit (rpoA)-binds DNA 1 x subunit (rpoB)- binds ribonucleotides 1 x ’ subunit (rpoC)-binds DNA 1 x subunit-rpoZ-RNAP assembly and transc. control 1 x subunit (rpoD, rpoH, rpoS and others)-binds -35 and -10 Sigma Factors •RNAP binds to promoters via interaction of the subunit with the -35 and -10 boxes. Sigma factors are elongated proteins that contact the -35 region with their d4 domain and the -10 region with the d2 (d2.4) domain. Alternative sigma factors recognize different promoters (Sigma and different promoter slides, RNAP bound to promoter) Gene: piece of DNA that encodes a protein or a functional RNA (e.g. tRNA or rRNA) Cistron: the coding region of a piece of DNA or RNA *Promoter: Binding site for RNA polymerase *Shine-Dalgarno site: Binding site for the 30S ribosomal subunit Open reading frame: Nucleic acid that does, or might encode a protein. It begins with a start codon (ATG, TTG, GTG) and ends with a stop codon (TAA, TAG and TGA) and is long enough to encode a protein (usually 50 amino acids). *Transcriptional terminator: place where RNA polymerase stops making RNA. *learn more about these shortly Features of bacterial mRNA •Prokaryotic RNA can be polycistronic (more than one gene can be found on a transcript). •Prokaryotic genes are often arranged in operons of genes encoding related functions. This allows coordinated regulation of these genes. •No intervening sequences (introns) have been found in eubacterial genes. Some archaeal tRNA genes and some phage genes have introns. •Transcription and translation are coupled in prokaryotes: translation occurs while the mRNA is being made. Translation Ribosome structure Ribosome structure slide 229 notes •rRNA operon structure: promoter-16S-tRNA-23S-5S-tRNA. These are excised by RNaseIII •Number of rRNA operons can vary from species to species, and there is a correlation between number and growth rate with fast-growing species having more. Species rRNA Growth rate Number Bradyrhizobium japonicum 1 10 hours Sinorhizobium meliloti 3 2.5 hours E. coli 7 20 minutes Vibrio natriegens 13 10 minutes tRNAs and the formation of aa-tRNA tRNA slides •tRNAs have two roles in translation: 1. They act as adaptors between mRNA and the ribosome. Ribosomes can’t decode mRNA without them. 2. They carry amino acids to the ribosome. •tRNAs need to be “charged with the proper amino acid. For example, tRNAgly needs to be charged with glycine. If it is mischarged with then the wrong a.a. will be incorporated into protein. REACTION (two steps each of which happens on the aa-tRNA-synthetase): 1. glycine + ATP > AMP-Glycine + PPi (reversible editing) 2. tRNAgly + AMP-Glycine tRNAgly-Gly + AMP •The correct aa-tRNA-synthetase must recognize both the a.a. and the tRNA. Note: there are: 20 a.a. x 20 tRNA types x 20 aa-tRNA synth = 203=24,000 possible combinations!! If done incorrectly (~23,999 ways to mess up), then the tRNA will be mischarged. (tRNA synthetase slide) •Charging is done by aminoacyl-tRNA-synthetases. Usually there is one for each amino acid. Therefore, all tRNAs for leucine are recognized by one aa-tRNAsynthetase which is specific for leucine and all of its tRNAs. •E. coli has 1 aa-tRNA-synthetase for each a.a., except lysine (it has 3) for a total of 22 aa-tRNA-synthetases tRNAs and genetic code •mRNA has to be decoded or translated by ribosomes which looks at groups of contiguous bases. how big are the groups? -Need to code for 20 amino acids -If one base was read, you could code for 41=4 amino acids (A,U,C,G) -If two bases were read, you could code for 42=16 amino acids (AA, AU, AC, AG…) -If three bases were read, you could code for 43=64 amino acids ENOUGH! Genetic code slide, CodonUsage slide Initiation •The 30S subunit initiates protein sythesis by binding at the start codon such that the initiator tRNA (tRNAfmet) is placed in the P site. This is true irrespective of the whether the start is AUG, GUG or UUG. ShineDalgarno •The first AUG codon on message is often NOT the start codon. So how does the ribosome find the true start condon? •The 3’ end of the 16S rRNA (in the 30S sunbunit) has a sequence that binds in an antiparallel and complimentary fashion to a seqence which is found 5-10 nt upstream of real start codons. The sequence on the mRNA (and which can be seen in the DNA) is the “Shine Dalgarno sequence. •When bound to the SD sequence, the AUG start codon is correctly positioned in the P-site of the 30S subunit. SD sequences slide Elongation •Release •When the ribosome reaches a stop codon, there is no tRNA with a compatible anticodon. Instead the codons are recognized by a protein release factor RF1 or RF2. RF1 recognizes UAA and UAG in the A-site RF2 recognizes UAA and UGA in the A-site •Ef-G and possibly RRF (ribosome release factor) bind and the peptide is cleaved from the peptide from the last tRNA which is in the P site. •RF3 binds and removes RF1 or RF2 from the ribosome. •The ribosome dissociates and the 30S subunit can bind a new mRNA •After release, the formyl group is usually removed from the f-met by a deformylase. •The reulting methionine is then often cleaved from the N-terminus of the peptide, if the next amino acid is small: ala, pro, ser, thr, gly, cys or val. Protein folding •No time to cover--read in text Gene of regulation expression Basic idea is to make only the proteins that are needed at any given time, and to make as many as are needed (not too few or too many) •Some proteins are needed all of the time and need to be synthesized continuously at the right rate: -ribosomes, cell wall synthesis enzymes, DNA synthesis enz. etc. •Some are needed only occasionally: -amino acid biosynthesis -proteins for degrading particular C-sources -virulence proteins Study of bacterial regulation of gene expression is a good model for intersting eukaryotic questions. (Slide) •Regulation of expression different places: occurs at a number of *=controlled here sometimes **=most common point of control Contol of transcription is the most important of these--most efficient to stop the process near the beginning rather than later. •best use of resources •not much sense in making proteins only to degrade them because they are not needed. Better to prevent their synthesis Hierarchry of Gene expression Single gene Operon (cluster of genes which share a promoter) Regulon: group of genes and/or operons which are controlled by a common regulatory protein. eg s32 Stimulon: a group of genes operons and/or regulons which are all controlled by a common environmental condition. E.G. the High Temperature Stimulon Two basic modes of transcriptional control •Negative control: •strong promoter, ground state is “on”. •Regulation is designed to keep it off when it is not needed (usually using a protein repressor that blocks RNAP) •Positive control: •weak promoter, ground state is “off” •regulation is designed to turn the promoter on when it is needed (usually by a protein activator that helps RNAP bind at the promoter, or that helps RNAP form an open complex) Positive control: the maltose regulon of E. coli Positive control by 2-component systems 2 parts, usually set up as follows: Membrane-bound sensor--called sensor kinase DNA binding protein, often a transcriptional activator Two component diagram Negative control Need a simple example here The DNA-binding proteins that effect positive and negative control recognize DNA binding sites through specific interactions between the protein and the DNA (usually in the major groove). The specificity give the activators and repressors the ability to recognize certain sequences--much like sigma factors recognize specific -35 and -10 regions. •A common protein motif that binds DNA is called “helix-turn-helix” -short 7 aa -helix + a 3-4 aa turn + 10-12 aa -helix -the longer helix sits in the major groove and provides much of the specificity -proteins with HTH often function as dimers and recognize their binding sites twice, in inverted repeats: LacI binding site O1: AATTGTGAGCGATAACAATT TTAACACTCGCTATTGTTAA (See slide repeats) for how protein dimers bind inverted •Pymol on LacI binding •Slides on other HTH proteins •SMART database search on LacI--use HTH to find all other HTHs in E. coli Monitoring Gene Expression