1 General Microbiology BIO30608 General Microbiology BIO306 Betül AKCESME MSc

2 Molecular Biology of ArchaeaChromosomes and DNA Replication in Archaea Transcription and RNA Processing in Archaea Protein Synthesis in Archaea Shared Molecular Features of Bacteria and Archaea

3 Bacteria vs Archea !!! no membrane-bound compartments and, in particular, no nucleus. both groups typically contain a single circular chromosome and often transcribe several genes onto the same polycistronic messenger RNA (mRNA). Comparative ribosomal RNA (rRNA) sequencing has revealed a closer relationship between Archaea and Eukarya than between Bacteria and Archaea.

4 Chromosomes and DNA Replication in ArchaeaChromosomes of Archaea resemble those of Bacteria DNA packaging and chromosome replication are more similar to Eukarya Archaea possess both DNA gyrase and histones Histones: eukaryotic-like proteins used for winding DNA DNA–histone structure known as nucleosome DNA gyrase, often referred to simply as gyrase, is an enzyme that relieves strain while double-stranded DNA is being unwound by helicase. This causes negative supercoiling of the DNA.

5 DNA+ histones =nucleosome 4 histones instead of 8 (as in eukaryotic cells) Archaeal histones are shorter in length than eukaryotic histones, but are homologous in amino acid sequence and similar in their three-dimensional structure. Central region of Histone is essential In bacteris , Histone like proteins are not homologous in sequence to true histones, nor do they form nucleosomes. Analogous histone-like proteins also occur in many Archaea. Archaea that grow at extremely high temperatures contain an enzyme called reverse DNA gyrase. This topoisomerase introduces positive supercoils into DNA. Reverse gyrase appears to play an important but undefined role in both Bacteria and Archaea that grow at extremely high temperatures.

6 Chromosomes and DNA Replication in ArchaeaArchaeal chromosome is circular like that of Bacteria and carrying from around 500 to a few thousand genes Replicates by bidirectional synthesis Machinery of replication is more similar to Eukarya The proteins of Archaea and Eukarya that recognize the origin of replication and help synthesize DNA the eukaryotic helicase forms rings consisting of six different protein subunits, whereas the archaeal helicase forms rings of six identical subunits. Archaea seem to have a simplified version of theeukaryotic replication apparatus. Has multiple replication origins (unlike bacteria)

7 All organisms possess multiple DNA polymerases specialized for different roles such as replication and DNA repair. Bacteria use DNA polymerases of family C as their main replicative enzymes, whereas DNA polymerases of family A and B are used mostly in DNA repair. In contrast, Archaea and Eukarya use DNA polymerases of family B as their main replicative enzymes

8 Transcription and RNA Processing in ArchaeaHave a simplified version of the eukaryotic transcription apparatus. Both the sequences of archaeal promoters and the structure and activity of RNA polymerase resemble those of eukaryotes. The regulation of transcription in Archaea shares major similarities with Bacteria! The Archaea contain only a single RNA polymerase Resembles eukaryotic polymerase II The antibiotic rifampicin inhibits bacterial RNA polymerase, but does not inhibit either the eukaryotic or archaeal enzymes The structure of archaeal promoters resembles that of eukaryotic promoters recognized by eukaryotic RNA polymerase II.

9     Bacteria Archaea EukaryaRNA polymerase from the three domains. RNA polymerase from the three domains.

10 DNA Promoter Start of transcription TBP TFB RNA polymeraseBinding of TBP and TFB Start of transcription TBP TFB Binding of RNA polymerase Promoter architecture and transcription in Archaea. Three promoter elements are critical for promoter recognition in Archaea: the initiator element (INIT), the TATA box, and the B recognition element (BRE). The TATA-binding protein (TBP) binds the TATA box; transcription factor B (TFB) binds to both BRE and INIT. Once both TBP and TFB are in place, RNA polymerase binds. RNA polymerase Transcription

11 Transcription and RNA Processing in ArchaeaEukaryotic genes have coding and noncoding regions Exons are the coding sequences Introns are the intervening sequences Are rare in Archaea Are found in tRNA and rRNA genes of Archaea Archaeal introns excised by special endonuclease Different then eukaryotes!

12 Primary transcript Mature (spliced) tRNA 3-exon 5-exon Splice siteBulge Splice sites Helix 5-exon Intron 3-exon Bulge Primary transcript Endonuclease cleavage Splice site 5-exon 3-exon P HO intron tRNA HO P Splicing of archaeal introns. Enzymatic ligation Intron 5-exon 3-exon Mature (spliced) tRNA tRNA precursor

13 Archaeal-style introns are also found in the nuclear tRNA genes of eukaryotesThe archaeal endoribonuclease that splices introns is homologous to two of the subunits of the enzyme complex that removes introns from eukaryotic nuclear tRNA.

14 Protein Synthesis in ArchaeaRemember phylogenetic tree!!!!! ( relationship between these three domain) Archaea share 67 of 78 proteins of the ribosome in common with Eukarya (34 are common for three domain) Eukarya and Archaea have twice as many translation factors as Bacteria Translational machinery is more similar between Eukarya and Archaea Both Archaea and Eukarya insert methionine as the first amino acid, in contrast to Bacteria, which use N-formylmethionine

15 Shared Features of Bacteria and Archaea !!!Bacteria and Archaea share several fundamental properties that are absent from eukaryotes Typically single-celled, most divide by binary fission Neither possesses a nucleus or membrane-bound organelles Archaea and Bacteria have coupled transcription and translation(no nucleus) Both possess a single, circular chromosome Both use Shine–Dalgarno sequences to indicate translation start The Shine-Dalgarno sequence (or Shine-Dalgarno box), proposed by Australian scientists John Shine (b.1946) and Lynn Dalgarno (b.1935),[1] is a ribosomal binding site in the mRNA, generally located 8 basepairs upstream of the start codon AUG. The Shine-Dalgarno sequence exists both in bacteria and archaea, being also present in some chloroplastic and mitochondial transcripts. The six-base consensus sequence is AGGAGG; in E. coli, for example, the sequence is AGGAGGU. This sequence helps recruit the ribosome to themRNA to initiate protein synthesis by aligning it with the start codon. The complementary sequence (CCUCCU), is called the anti-Shine-Dalgarno sequence and is located at the 3' end of the 16S rRNA in the ribosome. The eukaryotic equivalent of the Shine-Dalgarno sequence is called the Kozak sequence.

16 Eukaryotes alternate between haploid and diploid states.Both contain operon regulation of transcription in Archaea is largely bacterial in nature, relies on both repressor and activator DNA-binding proteins, which recognize sites within the promoter region Eukaryotes alternate between haploid and diploid states. In Bacteria and Archaea, however, reproduction and gene exchange are two distinct processes. There is no diploid phase, and reproduction is equivalent to cell division. RNA interference (RNAi), a major mechanism for protection against infection by RNA viruses, is found only in eukaryotes Bacteria and Archaea possess the CRISPR system of virus defense

17 Molecular features of the three domains!!!Archaea Eukarya Archaea Eukarya Bacteria Bacteria Genome Transcription and Translation 1 2 3 4 5 6 7 8 9 10 Chromosome circular versus linear Single chromosome versus multiple chromosomes Introns rare Archaeal-type introns Inteins Histones DNA gyrase Reverse gyrase Multiple chromosomal origins Eukaryotic origin recognition complex 11 12 13 17 18 19 Eukaryotic-type helicase B family DNA polymerase Is major replicative enzyme Eukaryotic-type sliding clamp Restriction enzymes RNAi Genome of double- stranded DNA Multiple retroelements in genome Centromeres Telomeres and telomerase 1 2 3 4 5 6 7 8 9 10 RNA used as a genetic messenger Polycistronic mRNA Cap and tail on mRNA TATA box and BRE sequence in promoter Repressors binding directly to DNA in promoter Multiple RNA polymerases RNA polymerase II with 8 or more subunits Multiple transcription factors needed Ribosomes synthesize proteins 70S versus 80S ribosomes 11 12 16 17 18 19 Ribosomal RNA sequence homologies Ribosomal protein sequence homologies Shine–Dalgarno sequences Multiple translation factors Elongation factor sensitive to diphtheria toxin N-Formylmethionine versus methionine tmRNA rescues stalled ribosomes 16S and 23S rRNA 18S, 28S, and 5.8S rRNA Figure 7.5 Molecular features of the three domains.

18 Eukaryotic Molecular BiologyGenes and Chromosomes in Eukarya Overview of Eukaryotic Cell Division Replication of Linear DNA RNA Processing Transcription and Translation in Eukarya RNA Interference (RNAi) Regulation by MicroRNA

19 Genes and Chromosomes in EukaryaPresence of nucleus has some circumstances! Cell division, transcription and translation Protein-encoding genes in Eukarya are often split into multiple exons (coding regions) by introns (noncoding regions) Both introns and exons are transcribed into the primary transcript Functional mRNA is formed by the excision of introns and splicing of exons

20 Primary RNA transcriptNucleus Gene A DNA Transcription Occurs in nucleus Primary RNA transcript RNA processing Cap and tail added Introns Mature mRNA Transport to the cytoplasm Cap Figure 7.6 Information transfer in eukaryotes. mRNA Occurs in cytoplasm Translation Poly(A) tail Protein A

21 Genes and Chromosomes in EukaryaEukaryotes contain much more DNA than is needed to encode all proteins required for cell functioning 3% of DNA in human genome encodes protein >90% of prokaryotic DNA encodes protein Eukaryotic microorganisms have fewer introns than higher eukaryotes Often have multiple copies of the same gene Have multiple linear chromosomes in the nucleus

22 Genes and Chromosomes in EukaryaEukaryotes wind DNA around histones to form nucleosomes Formation of the nucleosome introduce negative suprcoiling DNA–histone complex is called chromatin Highly condensed chromatin called heterochromatin Heterochromatin forms during eukaryotic cell division

23 Double-stranded DNA Nucleosome core Histone H1 Core histonesPackaging of eukaryotic DNA around a histone core to form a nucleosome. Packaging of eukaryotic DNA around a histone core to form a nucleosome. Nucleosomes are arranged along the DNA strand somewhat like beads on a string. In eukaryotes, nucleosomes consist of a core with eight proteins, two copies each of histones H2A, H2B, H3, and H4, plus a linker with one copy of histone H1. Core histones

24 Overview of Eukaryotic Cell DivisionHaploid and diploid! Single-celled eukaryotes, such as the brewer’s yeast, can exist indefinitely in the haploid stage (containing 16 chromosomes) as well as in the diploid stage (32 chromosomes). Occasionally, two haploid yeast cells will fuse (mate) to yield a diploid cell. (Different than higer eukaryotes!!) Mitosis Normal form of nuclear division in eukaryotic cells Chromosomes are replicated and partitioned into two nuclei Results in two diploid daughter cells in diploid cells and haploid in haploid eukaryotes! Meiosis Specialized form of nuclear division Halves the diploid number to the haploid number Results in four haploid gametes

25 Figure 7.8 Mitosis, as seen in the light microscope.Mitosis, as seen in the light microscope. These are onion root tip cells that have been stained to reveal nucleic acid and chromosomes. (a) Metaphase. Chromosomes are paired in the center of the cell. (b) Anaphase. Chromosomes are separating.

26 Replication of Linear DNAEukaryotic nuclei contain linear DNA Some viruses contain linear DNA Replication of DNA at extreme 5′ end represents a problem The problem is how to replace the RNA primer with DNA at the 5 end of the strand

27 Even if the RNA primer is very short and there is a special enzyme to remove it, no DNA polymerase can replace it with DNA because all known DNA polymerases require a primer. Therefore, if nothing was done about this problem, the DNA molecule would become shorter each time it was replicated. The replication of linear DNA thus requires special attention, and there are at least two solutions to this problem

28 Replication of Linear DNA Using a Protein PrimerViruses and plasimids which are linear Some DNA polymerases can add the first base onto an –OH group present on specific proteins that bind to the ends of linear chromosomes Recognize and bind the ends of the chromosomes. Not removed Replication of linear DNA using protein primers. Replication of linear DNA using protein primers. New strands of DNA are primed by proteins covalently attached to their 59 ends. Note the free –OH group on the protein. DNA polymerase III can add a nucleotide to this –OH group.

29 Replication of Linear DNAReplication of DNA at extreme 5′ end represents a problem Eukaryotes use telomeres and telomerase repetitive DNA—a short sequence (often 6 base pairs) tandemly repeated from 20 to several hundred times and Guanin rich During replication, this guanine-rich sequence is present on the leading strand of the DNA duplex and can base-pair with the 3 end of a complementary RNA molecule present in the enzyme telomerase Telomerase does not need a template to begin DNA synthesis

30 https://www.youtube.com/watch?v=AJNoTmWsE0s Leading strandTelomeric DNA Lagging strand RNA template Telomerase First 6-base extension Telomerase moves down one extension Model for the activity of telomerase at one end of a eukaryotic chromosome. Model for the activity of telomerase at one end of a eukaryotic chromosome. (a) A diagram of the sequence of the end of the DNA in a telomere with four of the guanine-rich repeats and the enzyme telomerase, which contains a short RNA template. (b) Steps in elongation of the guanine-rich strand catalyzed by telomerase. After telomerase finishes, the lagging strand can be primed with an RNA primer by primase, followed by completion of the lagging strand by DNA polymerase and ligase. (c) A preparation of HeLa cell chromosomes stained with fluorescent dyes. The red dots are leading-strand telomeres and the green dots are lagging-strand telomeres. Repeat four times https://www.youtube.com/watch?v=AJNoTmWsE0s RNA primer

31 Replication of Linear DNAEukaryotic chromosomes also contain centromeres Attachment site for spindle fibers      Spindle fibers pull the pairs of chromosomes apart during mitosis Kinetochore refers to the proteins that link the DNA of the centromeric region to the spindle fibers

32 Microtubules (spindle fibers)Outer kinetochore proteins CEN proteins Alpha satellite repeats Flanking repeats The eukaryotic centromere. The eukaryotic centromere. DNA at the centromere of human chromosomes consists of multiple repeats of 171 bp flanked by other repeats, both of which are highly condensed into heterochromatin. The centromere (CEN) proteins bind directly to the centromere DNA, and other proteins forming the kinetochore complex assemble onto the CEN proteins. The microtubules that make up the spindle attach to the kinetochore.

33 RNA Processing RNA processing: many RNA molecules are altered before they carry out their role in the cell RNA splicing Takes place in nucleus Removes introns from RNA transcripts Performed by the spliceosome (size like ribosome) RNA and Protein complex called called small nuclear ribonucleoproteins (snRNPs), Over 100 proteins participate to activitiy

34 Exported to cytoplasm for translationExon 1 Intron Exon 2 Conserved bases Assembly of spliceosome Spliceosome Cutting of 5 splice site, formation of lariat Cutting of 3 splice site, joining of exons Activity of the spliceosome. Activity of the spliceosome. Removal of an intron from the primary transcript of a protein-coding gene in a eukaryote. (a) A primary transcript containing a single intron. The sequence GU is conserved at the 59 splice site, and AG is conserved at the 39 splice site. There is also an interior A that serves as a branch point. (b) Several small ribonucleoprotein particles (shown in brown) assemble on the RNA to form a spliceosome. Each of these particles contains distinct small RNA molecules that take part in the splicing mechanism. (c) The 59 splice site has been cut with the simultaneous formation of a branch point. (d) The 39 splice site has been cut and the two exons have been joined. Note that overall, two phosphodiester bonds were broken, but two others were formed. (e) The final products are the joined exons (the mRNA) and the released intron. Intron excised Mature mRNA Intron (lariat) Exon 1 Exon 2 Degraded Exported to cytoplasm for translation

35 RNA Processing RNA processing Prior to splicing two other processings!Addition of methylated guanine to 5′ end of mRNA The guanosine cap is needed for translation and promotes the formation of the initiation complex between the mRNA and the ribosome Poly(A) tail Addition of 100–200 adenylate residues to 3’end the poly(A) tail is not translated Stabilizes mRNA and must be removed before the mRNA can be degraded. is required for translation, it indicates to the translation machinery that the RNA is mRNA rather than some other form of RNA and that it is ready for translation.

36 Pre-mRNA (primary transcript)Introns Poly(A) site Start Stop Exon 1 Exon 2 Exon 3 5-Cap 3-Polyadenylation Start Stop Poly(A) tail Occurs in the nucleus Introns excised Mature mRNA Processing of the primary transcript into mature mRNA in eukaryotes. Processing of the primary transcript into mature mRNA in eukaryotes. The processing steps include adding a cap at the 59 end, removing the introns, and clipping the 39 end of the transcript while adding a poly(A) tail. All these steps are carried out in the nucleus. The location of the start and stop codons to be used during translation are indicated. Start Stop Export to cytoplasm and translation Protein

37 RNA Processing Ribozymes: RNA molecules with enzymatic activityParticipate in several cellular reactions, the most important being polypeptide synthesis Ribozymes work like protein enzymes in Self-splicing intron: an intron that has enzymatic activity and splices itself out of RNA Most found in mitochondria and chloroplasts Also found in lower eukaryotes (e.g., Tetrahymena) Catalyze reaction only once Vestiges of simpler form of life?

38 rRNA precursor Mature rRNA Exon 1 Exon 2 Guanosine Intron (ribozyme)Excised intron HO rRNA precursor Mature rRNA Exon 1 Exon 2 Ribozyme circularizes HO 15-nucleotide fragment Self-splicing intron of the protozoan Tetrahymena. There is considerable secondary structure in such molecules, which is critical for the splicing reaction. (a) The rRNA precursor (primary transcript) contains a 413-nucleotide intron. (b) Following the addition of the nucleoside guanosine, the intron splices itself out and joins the two exons. (c) The intron is spliced out and then circularizes with the loss of a 15-nucleotide fragment. OH Exon splicing Degradation © 2012 Pearson Education, Inc.

39 Transcription and Translation in EukaryaEukaryotes have multiple RNA polymerases Unlike Bacteria and Archaea, which have just one. RNA polymerase I: transcribes genes for two large rRNA molecules, 18S and 28S RNA polymerase II: transcribes protein-encoding genes RNA polymerase III: transcribes genes for tRNA, 5S RNA, and other small RNA molecules Each class of RNA polymerase recognizes a distinct promoter (unlike prokaryotes)

40 DNA a few of the large number of accessory factors required forinitiation of transcription RNA poymerase II DNA a tail-like structure that can be phosphorylated, affecting activity of the polymerase The interaction of eukaryotic RNA polymerase II with a promoter. The interaction of eukaryotic RNA polymerase II with a promoter. The polymerase itself (brown) is positioned at the initiator element (INIT) of the promoter. A TATA-binding protein (yellow) is shown bound at the TATA box. The polymerase has a repetitive amino acid sequence at one end (shown as a tail-like structure) that can be phosphorylated, affecting activity of the polymerase. The other proteins shown in blue are a few of the large number of accessory factors required for initiation of transcription in eukaryotes. The interaction of eukaryotic RNA polymerase II with a promoter

41 Eukaryotic RNA polymerases require transcription factors to recognize specific promoters just as they do in Archaea. In both Archaea and Eukarya, general transcription factors are needed for the functioning of all promoters recognized by a particular RNA polymerase

42 Transcription and Translation in Eukarya !!!Protein synthesis is more complex in eukaryotes than in Bacteria Eukaryotic ribosomes are larger than bacterial ribosomes More initiation factors in Eukarya Eukaryotic mRNA is monocistronic Eukaryotic mRNA has no ribosome-binding site(Shine–Dalgarno sequence). Eukaryotic mRNA is recognized by its cap A specific protein, cap-binding protein, binds both the mRNA cap and the ribosome.

43

44 RNA Interference (RNAi !!!)Healthy cells do not contain dsRNA Presence of dsRNA indicative of RNA virus in cell RNA interference (RNAi): defense against dsRNA viruses Cleaves dsRNA Destroys ssRNA corresponding to targeted dsRNA sequence Found only in eukaryotes

45 RNA Interference (RNAi)RNAi triggered by dsRNA longer than 20 bp Long dsRNAs cleaved into 21–23 bp by Dicer nuclease Short interfering RNA (siRNA): 21–23 bp fragments from Dicer RNA-induced silencing complex (RISC): recognizes and destroys ssRNA corresponding to siRNA (by the nuclease Slicer, which is part of RISC) RNAi effect can travel through plasmodesmat (intercellular connections) in plants (copying of siRNA by an enzyme known as RNA-dependent RNA polymerase (RdRP)) RNAi effect can be passed through generations of Caenorhabditis elegans Mammals do not possess the RdRP needed to amplify RNAi, so the effect remains localized and cannot be transmitted.

46 dsRNA siRNA mRNA Dicer RISC Dicer cleaves dsRNA into shorter segmentsRISC binds siRNA and separates the strands RISC finds messenger RNA complementary to siRNA RNA interference. RNA interference. The nuclease Dicer cleaves doublestranded RNA into segments of 21–23 base pairs known as short interfering RNA (siRNA). This is recognized by RISC (RNA-induced silencing complex), which separates the strands of the siRNA. Finally, RISC cleaves target RNA that hybridizes to the siRNA. mRNA RISC cleaves mRNA RNA fragments degraded by exonuclease

47 Regulation by MicroRNAMicroRNAs (miRNAs): small dsRNAs that regulate translation in eukaryotic cells (unlike RNAi) Precursor folds to dsRNA Cleaved by Drosha (similar to Dicer) Trimmed by Dicer Bound to protein complex miRISC Binds to target on mRNA

48 DNA Nucleus RNA Cytoplasm miRNA mRNA Drosha Transcription FoldingCutting Export Cytoplasm Dicer Trimming miRNA Mechanism of miRNA action. Mechanism of miRNA action. The primary transcript for miRNA is made in the nucleus. The miRNA is cut out from the primary transcript by Drosha and trimmed further by Dicer after moving from the nucleus into the cytoplasm. The miRNA is then bound by miRISC, which separates the strands. One strand is used to base-pair with a target mRNA. This prevents translation of the mRNA. miRISC miRISC binding and strand selection mRNA Translation is blocked

49 miRNA tends to modulate the level of protein synthesis rather than switch it completely off.MicroRNAs are not only used by eukaryotic cells but also by some of the more complex viruses that infect them. Herpesviruses encode over 140 miRNAs