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Chapter 12

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

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Chapter 12

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  1. Chapter 12 Expression and Regulation

  2. Comparative GenomicsNCBICMR • 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

  3. 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

  4. 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

  5. Unique genes • ¼ of all genes are unique to a particular organism

  6. 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

  7. 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

  8. Gene expression • Transcription • Translation • Protein folding • Genes and gene regulation • Operons • Small RNAs

  9. 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 .

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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 packingwithin 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

  15. Core Enzyme Holoenzyme

  16. 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

  17. 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. Sigma factors

  18. 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

  19. 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

  20. 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

  21. Prokaryote Transcription

  22. Eukaryote Transcription

  23. 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 )

  24. 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

  25. Polysomes or polyribosomes • While RNA polymerase is synthesizing mRNA, the mRNA can already be attached to a ribosome • Protein synthesis can be initiated

  26. 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

  27. 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

  28. 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

  29. Site for attachment of the amino acid to the t- RNA • 3’ CCA – attaches to the terminal A

  30. Amino acyl tRNA synthetase • Amino acyl tRNA synthetase has two sites – one for the binding of the amino acid and a tRNA

  31. 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

  32. 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

  33. 16s rRNA

  34. E. coli ribosome Figure 12.13

  35. Ribosomal binding sites • P site or Donor site • A site or Acceptor site • E site or Exit site

  36. Initiation • Initiation in prokaryotes( Domain Bacteria) begins with a specially modified N- formylmeththionyl-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

  37. 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

  38. 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 bacterialmRNA. It is located 5' of the first coding AUG, and consists primarily, but not exclusively, of purines.

  39. 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.

  40. 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 N-formyl-methionyl-tRNA • The tRNA that matches this is for initiation only tRNAmet m

  41. 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

  42. 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.

  43. Elongation of the Polypeptide Chain • Every amino acid added to the growing polypeptide chain is the result of three phases • Amino acyl- tRNA binding • Transpeptidation reaction • Translocation

  44. 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.

  45. 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

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