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Chapter 12 Expression and Regulation Comparative Genomics NCBI CMR GC content – low of 29% for B. burdorferi to a high of 68% for M. tubercuolosis The difference in GC content affects the codon usage and amino acid composition for a species Glycine, alanine, proline, and arginine are represented by GC rich genomes. Isoleucine, phenylalnine, tyrosine, and methionine are represented by AT rich codons Shared genes ½ of all genes are similar or homologous in bacterial species The number of genes involved in processes like transcription and translation are similar even when there is a vast difference in the size of genomes Suggestive of a basic number for all processes in the cell Transport Genes A high number of transport genes required to move molecules across a membrane Genome size and the different transport mechanisms are related Many transport systems are based on the life style, for instance heterotrophy Unique genes ¼ of all genes are unique to a particular organism Evolution Vertical transmission Duplication of genes after vertical transmission Horizontal or lateral transmission of unique genes Conjugation, transformation, and transduction( phages) Pathogenicity islands – blocks of pathogenic genes transferred with selective advantage Molecular evidence BLAST – similarity in genes by homology and alignment COG – Clusters of orthologous groups, classifies genes on the basis of similar function Ribosomal genes and small RNA’s Whole genome analysis Gene expression Transcription Translation Protein folding Genes and gene regulation Operons Small RNAs Prokaryote mRNA Ss RNA( 5’ ---------3’) Directions for one or more polypeptides Non translated leader sequence of 24 to 150 bases at the 5’ end Polygenic RNAs that code for more than one polypeptide have spacers At the 3’ end following the termination codon there is a non translated trailer . RNA Polymerase RNA is synthesized under the direction of RNA polymerase The synthesis is similar to that of DNA Nucleotide tri-phosphates n[ ATP,GTP,CTP,UTP] RNA+ nPPi Pyrophosphate( PPi ) Pyrophosphate is produced in both DNA and RNA Polymerase reactions Pyrophosphate is then removed by hydrolysis to orthophosphate in a reaction catalyzed by the phosphatase enzyme The reaction is irreversible RNA polymerase The RNA polymerase of E. coli is an extremely large enzyme It contains four polypeptide chains The RNA polymerase opens or unwinds the double helix to form a transcription bubble about 12 – 20 base pairs in length It transcribes the mRNA from 5’ to 3’ It produces mRNA at about 40 nucleotides/second at 37oC. Core enzyme component Catalytic activity Composed of four chains The Sigma factor has no catalytic activity but assists in the recognition of genes. Once transcription begins this factor dissociates from the core enzyme complex The Beta and Beta prime polypeptides are involved with the ginding of DNA and regulation. Rifampin which is a polymerase inhibitor binds to the B’ The function of the Alpha subunit is involved in the recognition of the promoters RNA Polymerase These are the different views of the core RNA polymerase molecules as they observed on the surface of a lipid bilayer tube. Each picture shows three molecules which appear linked. It happens because negative stain does not penetrate between the molecules due to their tight packing within a helical crystal. The most striking feature of the core structure is a thumb-like projection surrounding a channel. The channel is 25 Å in diameter and can easily accommodate double stranded DNA. For more information on this Core Enzyme Holoenzyme Sigma Factors Sigma70 Primary sigma factor, or housekeeping sigma factor. Encoded by rpoD . When bound to RNAP Core allows recognition of -35 and -10 promoters. No other factors required for RNAP binding and transcription initiation. Sigma54 alternative sigma factor involved in transcribing nitrogen-regulated genes (among others). Encoded by rpoN (ntrA ). When bound to RNAP Core allows recognition of different -26 and -12 promoters. Requires an additional activator to allow open complex formation for transcription. Sigma factors Sigma32 heat shock factor involved in activation of genes after heat shock. Encoded by rpoH (htpR ). Turned on by heat shock (either at the transcription or protein level). Activates multiple genes involved in the heat shock response. SigmaS (sigma38) Stationary phase sigma factor. Encoded by rpoS . Turned on in stationary phase. Activates genes involved in long term survival, peroxidase. RNA Binding Binding occurs with the aid of the Sigma factor Recognition site is TTGACA about 35 bases upstream of the gene The TATAAT sequence or Pribnow box lies within the promoter about 10 base pairs before the starting point of transcription. RNA polymerase recognizes these sequences The DNA begins to unwind near the Pribnow box Transcription begins about 6 or 7 base pairs from the 3’ end of the promoter Thermus aquaticus – RNA polymerase The enzyme is composed of four subunits and is complexed with the sigma factor The structure is claw shaped. Has an internal channel that contains Mg++ This may provide an entry point for DNA The sigma unit binds to the -10 and -35 elements of the promoter Termination There should be a stop codon And there must also be signals for termination Terminator often contain a sequence coding for an RNA stretch that can form a hair pin with complementary base pairing This works as a signal for RNA polymerase to stop transcription Prokaryote Transcription Eukaryote Transcription Protein Synthesis The mRNA is translated into the amino acid sequence of a protein In E. coli protein synthesis is rapid and accurate. It occurs at a rate of 900 residues per minute The synthesis of a polypeptide chain begins at he free amino group end( N- terminus) and concludes with the carboxyl group at the end( the C- terminus ) Bacterial translation To account for the rapid growth of bacteria, m RNAs must be used efficiently They can complex with several ribosomes at a time There may be a ribosome every 80 nucleotides on the mRNA and as many as 20 ribosomes reading the mRNA transcript These complexes are called polyribosomes Polysomes or polyribosomes While RNA polymerase is synthesizing mRNA, the mRNA can already be attached to a ribosome Protein synthesis can be initiated tRNA – Clover leaf – loops and stem CCA terminus( 3’ ) attachment for amino acid Anticodon at the base 3’-----5’ Three letters complementary to mRNA sequence There are two large arms : the D arm has a substitution of a pyrimidine nucleotide – dihydrouridine tRNA is folded into an L-shaped structure. The amino acid is held on one end of the L Attachment of an amino acid to a tRNA This is called Amino Acid activation The tRNAs are approximately 73-93 nucleotides in length The acceptor end of the tRNA ends in C-C- A. ( 3’ end) The amino acid attaches to the terminal adenylic acid Attachment of an amino acid to a tRNA The attachment of an amino acid to a tRNA is catalyzed by an enzyme called aino-acyl-tRNA synthetase The association of the amino acid and the tRNA requires the use of ATP in the presence of a Mg++ There are at least 20 amino acyl tRNA synthetases Site for attachment of the amino acid to the t- RNA 3’ CCA – attaches to the terminal A Amino acyl tRNA synthetase Amino acyl tRNA synthetase has two sites – one for the binding of the amino acid and a tRNA Prokaryote ribosome Prokaryote ribosomes consist of a 30 s and a 50 s subunit Each subunit is composed of one or two rRNA molecules and many proteins The total complex is 70s Prokaryote Ribosomes Ribosomal RNA has three roles The 16 s rRNA of the 30 s portion of the ribosomes may aid in the initiation of protein synthesis It can bind to the initiation factors It may also have a catalytic function 16s rRNA E. coli ribosome Figure 12.13 Ribosomal binding sites P site or Donor site A site or Acceptor site E site or Exit site Initiation Initiation in prokaryotes( Domain Bacteria) begins with a specially modified Nformylmeththionyl-tRNA This molecule binds to the 30s sub unit of the ribosome and is possitioned with both the 3’ end and the 16srRNA and the anticodon of the fMet-tRNA Messengers have a special initiator codon 5’ AUG or GUG that specifically binds with the fMet- tRNA Initiation Factors( associated with the 30s subunit) Three initiation factors are required IF-3 promotes the binding of the mRNA to the 30s unit( also stabilizes the binding) IF-2 binds GTP and fMet-tRNA and the 30s unit IF-1 is needed for the release of IF-2 and GDP from the reaction which requires the use of a phosphate for energy IF3 IF3 recognizes the sequence of the ribosome binding site on the bacterial m RNA. This is called the Shine-Dalgarno sequence. AGGAGGU) is the signal for initiation of protein biosynthesis in bacterial mRNA. It is located 5' of the first coding AUG, and consists primarily, but not exclusively, of purines. Shine-Dalgarno The requirement for a Shine-Dalgarno sequence in addition to AUG for proper initiation allows the AUG to be chosen from among multiple AUG trinucleotides in mRNA, most coding for internal methionines or representing out of phase codons. Binding of mRNA to rRNA via the Shine Dalgarno sequence may stimulate initiation by increasing the local concentration of AUG near the correct site on the ribosome. Other sequences, in addition to the AUG and Shine-Dalgarno sequence, are also important. Initiation codon AUG GUG UUG In bacteria the initiator tRNA carries a methionine residue that has been formylated on its amino group forming a molecule of Nformyl-methionyl-tRNA The tRNA that matches this is for initiation only tRNAmet m Initiation The AUG at the start position in mRNA codes for formyl methionine The AUG in other positions codes for methionine The translation of AUG and GUG depends upon the context Translation In bacteria and mitochondria,the formyl residue is removed by a specific deformylase enzyme to generate a normal NH2 terminus. If methionine is to be the NH2 terminal amino acid this is the only step. In about ½ of the proteins aminopeptidase removes the methionine creating a new terminus. Elongation of the Polypeptide Chain a. b. c. Every amino acid added to the growing polypeptide chain is the result of three phases Amino acyl- tRNA binding Transpeptidation reaction Translocation Elongation factors GTP and the elongation factor EF-Tu are required for the insertion of the first t-RNA into the A site( EF-Tu is associated with the ribosome. This is followed by GTP hydrolysis and the GDPTu complex leaves the ribosome EF-Tu.GDP is converted to EF-Tu.GTP with the aid of a second elongation factor EF-Ts. GTP – The entry of the amino acyl t- RNA to the A site is dependent upon a guanine nucleotide When GTP is present, the factor is in its active state When the GTP is hydrolyzed to GDP, the factor becomes inactive Activity is restored when the GDP is replaced by GTP Elongation cycle At the beginning of the elongation cycle the peptidyl site is filld with either Nformylmethionyl-tRNAor peptidyl-tRNA and the A and E sites are empty The second amino acyl-tRNA is inserted into the A site Transpeptidation Occurs between amino acids This is catalyzed by peptidly transferase located on the 50s subunit The two amino acids are joined by a peptide bond No extra energy source is required Transpeptidation reaction catalyzed by peptidyl transferase Figure 12.16 Transpeptidation factors Transcription elongation factors stimulate the activity of the RNA polymerase by increasing the overall elongation rate and the completion of RNA chains. E. coli GreA is one such factor. It acts by inducing cleavage of the transcript within the RNA polymerase, followed by release of the RNA 3'-terminal fragment. Translocation The final stage of the elongation process of protein synthesis The peptidyl – tRNA moves about 20 Angstroms from the A to the P site The ribosome moves one codon along the mRNA so that a new codon is positioned on the A site The empty rRNA leaves the ribosome Ribosomal proteins are involved in this movement Translocation the final step in elongation The three aspects of this process occur simultaneously Translation and Moving Molecules faculty.smu.edu/ svik/5304/molecules.html Termination of Translation in Prokaryote Protein synthesis stops when a nonsense codon. UAA, UGA, UAG reaches the ribosome Three release factors are required GTP hydrolysis is required The ribosomal subunits dissociate from each other and the mRNA is released Antibiotics that affect the process of translation Kirromycin inhibits the function of EFTu Puromycin mimics amino acyl t RNA Erythromycin blocks peptidyl transferase Streptomycin blocks initiation Protein folding and chaperones Molecules called chaperone recognize only unfolded polypeptides or partially denatured proteins They suppress incorrect folding and promote correct folding to achieve the conformation of the tertiary structure and shape of the protein Bacterial Chaperones a. b. Best studied in E. coli Four chaperons are invovlved DnaK, DnaJ,GroEL, and GroES Also the stress protein GrpE Protein splicing in prokaryotes Some microbial proteins are spliced after translation In microbial splicing a part of the polypeptide is removed before it folds into its final shape Self-splicing proteins are large and have internal intervening sequences called inteins ( 130-600 bases in length) flanked by external sequences exteins. Inteins are removed by an autocatalytic process. Protein splicing in prokaryotes Removal of part of polypeptide before folding Inteins – removed portion Exteins – portions that remain in protein Prokaryote vs. Eukaryote folding Domains – structurally independent regions of polypeptide – separated from each other by less structured portions of polypeptide In eucaryotes – domains fold independently right after being synthesized – molecular chaperones not as important In procaryotes – polypeptide does not fold until after synthesis of entire polypeptide – molecular chaperones play important role Chaperone Action DnaJ binds to the unfolded chain DnaK is complexed with ATP and attaches to the polypeptide to prevent improper folding as it is synthesized The ATP is hydrolyzed after binding GrpE binds to the chaperone-polypeptide complex and causes the release of ADP and DnaJ and K are also released from the polypeptide Often GrolEl abd GroEs will be involved in the final folding They receive the protein from DnaJ and DnaK This process also requires the hydrolysis of ATP Prokaryote folding and splicing A part of the protein is removed before folding Inteins are about 130-600 amino acids in length are removed in an autocatalytic process This is a relatively new discovery – examples include the RecA protein in Mycobacterium tuberculosis ( Bacteria)and the DNA polymerase in Pyrococcus( Archaea). The presence of these self splicing proteins in Bacteria and Archaea suggest that this principle is wide spread. Chaperone activity Regulation of mRNA synthesis The control of metabolism by the regulation of enzyme activity is a necessary means of control in a unicellular entity. The need to control gene expression is vital to their ability to adjust to changing environmental conditions It is also necessary to conserve energy by only expressing those genes that are necessary at any moment in time for survival under a set of conditions. Induction and repression Inducible enzymes are those that are produced as a result of the presence of a small molecule called an inducer They are used only in the presence of their substrate Repressible enzymes are those that are regulated by the end product of the reaction. Repressible enzymes are regulated by the formation of their product which acts to slow their production Control Negative control – A controlling factor can either inhibit or activate transcription. Both induction and repression are forms of negative control. mRNA synthesis proceeds more rapidly in the absence of the controlling factor. Control II The rate of mRNA synthesis is controlled by special repressor proteins that are synthesized under the direction of regulator genes. The repressor binds to a special site on the DNA called the operator. The inactivation of the regulatory gene produces a constitutive mutant – in which mRNA synthesis occurs whether the repressor is present or absent Inducible systems The regulator gene directs the synthesis of an active repressor The inducer stimulates transcription by binding to the repressor causing it to change to an inactive shape. Repressible systems The repressor is initially in an inactive form called the aporepressor. The aporepressor becomes active only when a corepressor binds to it The corepressor inhibits transcription by activating the aporepressor Regulation of biosynthesis The synthesis of genes for a pathway can be sequentially arranged in the DNA There may be only one repressor to regulate the action of the structural genes coding for a polypeptide. A single messenger RNA will contain the genetic code for all the proteins in the pathway Operon The sequence of bases coding for one or more polypeptides together with the operator that controls its expression is called an operon Lac Operon Works by negative control Contains three structural genes Controlled by the lac repressor Beta galactosidase Beta galactoside permease Beta galactoside transacetylase Beta galactosidase Reaction The lac operon is necessary for the metabolism of the sugar, lactose Negative control The inducer can bond to the repressor and inactivate it When this occurs the genes are transcribed promoter usually substrate of pathway operator negative control of catabolic pathway an operon structural gene = gene coding for polypeptide Lactose repressor The lactose repressor binds to the DNA and prevents the transcription of the three structural genes The lac Operon is also under postive control It is regulated by CAP or catabolite activator protein or cyclic AMP receptor protein and the small cyclic nucleotide 3’,5’- cyclic adenosine monophosphate cAMP or cyclic AMP CAP recognize and bind regulatory region of lactose operon Repressor and CAP bound to the lac operon The lac operator is violet The operators are red The promoters are green, CAP is blue In this conformation there is no transcription CAP Absence of the lac repressor is essential but not sufficient for effective transcription of the lac operon. The activity of RNA polymerase also depends on the presence of another DNA-binding protein called catabolite activator protein or CAP. Like the lac repressor, CAP has two types of binding sites: One binds the nucleotide cyclic AMP; the other binds a sequence of 16 base pairs upstream of the promoter CAP However, CAP can bind to DNA only when cAMP is bound to CAP. so when cAMP levels in the cell are low, CAP fails to bind DNA and thus RNA polymerase cannot begin its work, even in the absence of the repressor. Structure of CAP Two recognition sequences Recognition sequences are 34 A apart CAP binding CAP strategy They usually contain two subunits. Therefore, they are dimers. They recognize and bind to DNA sequences with inverted repeats. In prokaryotes, recognition and binding to a particular sequence of DNA is accomplished by a segment of alpha helix. Hence these proteins are often described as helix-turn-helix proteins Catabolite repression Catabolite repression of lactose and other operons by glucose – glucose decreases cAMP levels, thereby blocking CAP binding and decreasing mRNA synthesis – When glucose is present, the cAMP level decreases and the lac operon is inhibited – The decrease in cAMP may be due to the effect of the PTS system on the activity of adenyl cylase, the enzyme that sytnesizes cAMP Attenuation Bacteria can regulated transcription in an alternative manner An example of this is the tryptophan operon in E. coli. The operon which contains the code for five structural genes is under the control of a repressor protein TrpR gene The trp gene codes for the repressor protein Excess tryptophan inhibits transcription of the operon genes by acting as a corepressor and activating the repressor protein. Attenuation A leader region lies between the operator and the first structural gene in the operon The trp gene is responsible for controlling the continuation of transcription after the RNA polymerase has bound to the promoter The leader region contains an attenuator and a sequence that codes for the leader peptide The attenuator is a rho independent termination site – It is GC rich followed by eight uridine residues The residues can pair with each other to form hairpin loops. In the absence of a ribosome, the loops are formed and transcription will terminate Tryptophan operon http://science.nhmccd.edu/biol/operon/ toff.html Attenuation continued If When tryptophan is present, there is sufficient tryptophanyl-tRNA for protein synthesis – therefore the leader peptide will continue moving along the mRNA until it reaches a UGA stop codon, at which time will form hairpin loops with complementary base pairing Ribosome behavior influences translation of the mRNA as it regulates the RNA polymerase activity. Five other amino acid pathways have similar means of regulation Please refer to diagram in booklet Attenuation I A leader region lies between the operator and the first structural gene in the operon the trpE. It is responsible for controlling the continuation of transcription after the RNA polymerase has bound to the promoter the tryptophan operon leader of operon High and Low Tryptophan levels Tryptophan Operon Arabinose operon Arabinose products The ara operon codes for three enzymes that are required to catalyze the metabolism of arabinose. Arabinose isomerase - encoded by araA - coverts arabinose to ribulose Ribulokinase - encoded by araB -- phosphorylates ribulose Ribulose-5-phosphate epimerase - encoded by araD -- converts ribulose-5-phosphate to xylulose-5-phosphate which can then be metabolized via the pentose phosphate pathway. Arabinose operon araO1 is an operator site. AraC binds to this site and represses its own transcription from the PC promoter. In the presence of arabinose, however, AraC bound at this site helps to activate expression of the PBAD promoter. araO2 is also an operator site. AraC bound at this site can simultaneously bind to the araI site to repress transcription from the PBAD promoter araI is also the inducer site. AraC bound at this site can simultaneously bind to the araO2 site to repress transcription from the PBAD promoter. In the presence of arabinose, however, AraC bound at this site helps to activate expression of the PBAD promoter. CRP binding site CRP binds to the CRP binding site. It does not directly assist RNA polymerase to bind to the promoter in this case. Instead, in the presence of arabinose, it promotes the rearrangement of AraC when arabinose is present from a state in which it represses transcription of the PBAD promoter to one in which it activates transcription of the PBAD promoter. Arabinose absent When arabinose is absent, there is no need to express the structural genes. AraC does this by binding simultaneously to araI and araO2. As a result the intervening DNA is looped. These two events block access to the PBAD promoter which is, in any case, a very weak promoter (unlike the lac promoter): Arabinose present When arabinose is present, it binds to AraC and allosterically induces it to bind to araI instead araO2. If glucose is also absent, then the presence of CRP bound to its site between araO1 and araI helps to break the DNA loop and also helps AraC to bind to araI: Global Regulatory Systems Affect many genes and pathways simultaneously Regulon – collection of genes or operons controlled by a common regulatory protein – This enhances the cells ability to coordinate cellular processes Asn C transcriptional regulator ATGGAAAATT ATCTGATCGA CAATCTGGAC CGTGGCATCC TGGAAGCATT AATGGGCAAT GCGCGCACCG CTTACGCCGA ACTGGCGAAA CAATTTGGCG TCAGTCCGGG GACGATTCAC GTTCGAGTAG AGAAAATGAA GCAGGCGGGG ATCATTACCG GGGCGCGTAT TGATGTCAGC CCGAAGCAGC TCGGTTATGA CGTAGGCTGC TTTATCGGCA TTATATTAAA GAGCGCCAAA GACTACCCTT CCGCGCTGGC AAAGCTGGAA AGCCTTGATG AAGTCACTGA AGCCTATTAC ACAACCGGCC ACTACAGCAT CTTTATAAAA GTGATGTGCC GTTCGATCGA CGCTCTCCAG CATGTACTTA TCAACAAGAT CCAAACAATT GATGAAATTC AGTCCACCGA GACATTGATC GTCCTGCAAA ACCCGATCAT GCGTACCATC AAGCCCTGA Global regulatory Heat shock – chaperones that respond to elevations in temperature – If the cell temperature is too highthe amount of heat shock proteins increases SOS repair system – in which the damage to DNA is so extreme that it utilizes ( Rec system) Catabolite Repression Occurs when operon is under control of catabolite other than initial substrate of pathway Allows preferential use of one carbon source over another when both are available in environment E. coli preferentialloy uses glucose – when the glucose supply is exhausted, the bacterium can switch to lactose Regulation by Sigma Factors and Control of Sporulation Different sigma factors recognize different promoters Substitution of sigma factors changes gene expression of many genes and operons Bacillus subtilis sporulation sigma factors Synthesized only as cell switches from vegetative growth to sporulation Lead to transcription of sporulation-related genes Small RNAs (sRNAs) and Regulation Also called noncoding (nc)RNAs Do not function as mRNA or rRNA There are between 50 and 200 of these In E. coli there may be as many as 50-400 nucleotides in length . Appear to regulate genes by three different mechanisms – Pair directly with other RNAs via RNA-protein interactions (e.g., OxyS RNA) – Intrinsic activities (e.g., RNase P RNA and tmRNA) – Antisense RNA has a base sequence complementary to a segment of another RNA and preferentially binds to this, inactivating it OxyS RNA of E. coli Made in response to hydrogen peroxide exposure Can act as an antisense RNA – binds directly to mRNA and blocks translation Can also block translation by binding a protein required for translation of a target mRNA micF RNA of E. coli Regulates synthesis of OmpF porin protein – porin proteins are outer membrane proteins – different porins produced under different conditions OmpC porin made when in intestine OmpF porin made when in dilute environment MicF antisense RNA binds OmpF RNA and blocks its translation when bacterium in intestines RNase P RNA the RNA component of RNase P has catalytic activity responsible for tRNA processing tmRNA of E. coli Helps repair problems caused by defective mRNAs that lack stop codons Acts as both alanyl-tRNA and mRNA when ribosome stalls at end of defective mRNA Two functions – releases ribosome from defective mRNA – adds carboxy-terminal polypeptide tag to protein, marking it for degradation Two-Component Phosphorelay Systems Transfer of phosphoryl groups- control gene transcription and protein activity Signal transduction situation Consists of a sensor kinase And A response regulator Two examples sporulation and chemotaxis Sporulation continued SpoOF donates the phosphoryl group to a histidine on SpoOB. SpoOA is a response regulator It has a receive domain aspartate and picks up the phosphoryl group from SpoOB to become an active transcription regulator. Sporulation in B. subtilis Figure 12.33 Chemotaxis in E. coli Figure 12.34 Control of the Cell Cycle Cell cycle – complete sequence of events extending from formation of a new cell through next division – requires that DNA replication and cell division be tightly coordinated Precise mechanisms of control are not known Cell cycle control in E. coli two separate control pathways – sensitive to cell mass – sensitive to cell length Figure 12.36 Effect of growth rate slow growth rate – DNA replicated then septation begins rapid growth rate – DNA replicated and new round of DNA replication begins before septation begins Figure 12.35