Information Flow in Microbes Part 2
- 1. Molecular Information Flow in Microorganisms
- 2. Gene, Genetic Elements, and Genomes • The functional unit of genetic information is the gene • Genes make up parts of chromosomes or other large molecules known collectively as genetic elements • The total complement of genetic elements is the genome (chromosome)
- 3. Central Dogma DNA begets RNA begets Protein
- 4. Central Dogma 1. Replication. During replication, the DNA double helix is duplicated. Replication is catalyzed by the enzyme DNA polymerase. 2. Transcription. The transfer of genetic information from DNA to RNA. Transcription is catalyzed by the enzyme RNA polymerase. 3. Translation. The formation of a polypeptide using the genetic information transferred to mRNA by DNA is a process that occurs on the ribosome.
- 5. Central Dogma • In Eukaryotes, replication and transcription occur in nucleus (because no ribosomes there). • In Bacteria and Archaea, it all happens in cytoplasm, and a ribosome initiates translation of an mRNA before RNA polymerase has finished synthesizing it. • Allows for rapid cell growth
- 7. Chromosomes • Bacteria and Archaea (mostly) have a single, circular chromosome. • Contains all (or most) of the organism’s genes • Eukaryotes have (multiple), linear chromosomes.
- 8. Plasmids • Many Bacteria and Archaea contain plasmids in addition to their chromosome • Thousands of different plasmids are known • Most plasmids are circular, but many are linear and vary in size from approximately 1 kbp to more than 1 Mbp
- 9. Plasmids • Pathogenic bacteria express a variety of plasmid-encoded virulence factors that assist them in establishing infections. • Sometimes plasmids encode properties essential to the ecology of the bacterium. • Nitrogen fixation in Rhizobium • Genes for degradation of hydrocarbons or toxic pollutants, such as polychlorinated biphenyls (PCBs) and herbicides or other pesticides
- 10. DNA Replication • Replication is a semiconservative process • The two resulting double helices each consist of one new strand and one parental strand • DNA replication always proceeds from the 5’ end to the 3’ end • the 5’-phosphate of the incoming nucleotide being attached to the 3’-hydroxyl of the previously added nucleotide
- 11. DNA Replication • Enzymes that catalyze the polymerization of deoxynucleotides are called DNA polymerases • All DNA polymerases synthesize DNA in the 5’ to 3’ direction, but none of them can initiate a new chain de novo • They can only add a nucleotide onto a preexisting 3’-OH group • To start a new DNA chain, a primer is required • A nucleic acid molecule to which DNA polymerase can attach the first nucleotide
- 12. Transcription • Transcription transforms the genetic information inscribed in DNA into RNA • Yields three main forms of RNA: • messenger (mRNA), • transfer (tRNA), • ribosomal (rRNA)
- 13. Transcription in Bacteria • Transcription is catalyzed by the enzyme RNA polymerase • During elongation of an RNA chain, ribonucleoside triphosphates are added to the 3’-OH of the ribose of the preceding nucleotide. • Thus chain growth is 5’ to 3’, just as in DNA synthesis
- 14. Transcription in Bacteria • RNA polymerase uses DNA as a template • For any given gene, only one of the two strands is transcribed • Unlike DNA polymerase, RNA polymerase can initiate new RNA on its own • Unlike DNA replication, transcription occurs on much smaller units of DNA, often as little as a single gene Cryo-electron micrograph of Escherichia coli RNA polymerase with sigma factor
- 15. Transcription in Bacteria • Gene expression is a highly regulated process • To begin transcription, RNA polymerase must first recognize initiation sites on the DNA • Sites are called promoters • The DNA helix at the promoter site is opened up by RNA polymerase • As the polymerase moves, it unwinds the DNA in short segments to expose template DNA
- 16. Transcription in Bacteria • Promoters are specific DNA sequences • In Bacteria, promoters are recognized by sigma. • Sigma recognizes two highly conserved regions within the promoter These sequences are recognized by the same E. coli sigma factor called 𝜎70
- 17. Transcription in Bacteria • Genetic information is organized into transcriptional units • Segments of DNA that are transcribed into a single RNA molecule bounded by their initiation and termination sites. • Can be: • A single gene • Multiple genes • An operon is a unit of two or more genes transcribed into a single RNA, and under the control of a single regulatory site (promotor)
- 18. Transcription in Bacteria • Termination of transcription is governed by specific base sequences on the DNA. • In Bacteria a common termination signal is a GC- rich sequence containing an inverted repeat. • When this DNA sequence is transcribed, the RNA forms a stem–loop structure by intra- strand base pairing
- 19. Translation in Bacteria • Once transcription has occurred, the mRNAs are translated into protein • Transfer RNAs function to carry amino acids to the translation machinery • Each tRNA contains a specific three-nucleotide sequence called the anticodon • Group of three bases that recognizes a codon (a three-base code for an amino acid) on the mRNA
- 20. Translation in Bacteria • The correct amino acid (aka. cognate amino acid) is linked to a specific tRNA by an enzyme called an aminoacyl-tRNA synthetase • For each amino acid, a separate aminoacyl-tRNA synthetase exists • Specifically binds both the cognate amino acid and the tRNA containing the corresponding anticodon
- 21. Translation in Bacteria • An mRNA triplet of three bases, called a codon, encodes each specific amino acid • Codons themselves are encoded by the organism’s genome • There are 64 possible codons (4 DNA bases taken 3 at a time = 43) • In addition to the codons for amino acids, there are also codons for starting and stopping translation • There are 22 naturally occurring amino acids • Several amino acids can be encoded by more than one codon • Called degenerate code
- 22. Translation in Bacteria • A codon is recognized by specific base pairing with a complementary sequence on the anticodon, located on a tRNA • If this base pairing were always the standard pairing of A with U and G with C, then at least one specific tRNA would be needed to recognize each codon. • True for leucine in E. coli • Not true for lysine • One tRNA that recognizes AAA or AAG • This phenomenon is called wobble
- 23. Translation in Bacteria • Messenger RNA is translated beginning with its start codon (AUG) • Encodes a chemically modified methionine in Bacteria called N-formylmethionine • With triplet code, its important that a peptide sequence starts in the right place. • The reading frame that when translated yields the polypeptide encoded by the gene is called the 0 (zero) frame • In Bacteria, ribosomal RNA recognizes a specific AUG on the mRNA as a start codon
- 24. Translation in Bacteria • If an mRNA can be translated, it is because it contains an open reading frame (ORF) • A start codon followed by a number of codons and then a stop codon in the same reading frame as the start codon. • The codons UAA, UAG, and UGA are stop codons • Using computational methods, a DNA base sequence can be scanned to search for open reading frames. • The search for ORFs is central to the field of genomics
- 25. Translation in Bacteria • Ribosomes are the sites of protein synthesis. • Bacteria and Archaea have 30S and 50S ribosomal subunits that yield intact 70S ribosomes. • The 30S subunit contains 16S rRNA and 21 proteins, and the 50S subunit contains 5S and 23S rRNA and 31 proteins. • In Bacteria, initiation of protein synthesis begins with a free 30S ribosomal subunit • Next, a 50S ribosomal subunit is added to the initiation complex to form the active 70S ribosome.
- 26. Translation in Bacteria • During translation, the mRNA threads through the ribosome bound to the 30S subunit • The A site is where the incoming charged tRNA first attaches. • The P site is where the growing polypeptide chain is attached to the prior tRNA • During peptide bond formation, the growing polypeptide chain moves to the tRNA at the A site • Following elongation, the tRNA holding the polypeptide is translocated from the A site to the P site, opening the A site for a new charged tRNA. • Translocation pushes the now amino acid–free tRNA to a third site, called the E (exit) site • It is from here that the tRNA is released