Bacterial Genetics: DNA Replication, Transcription, and Translation, Study notes of Microbiology

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Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy Microbiology Test 3 Genomes and Chromosomes Chapter Overview:

- DNA 101 - Genetic code: BIG picture - Microbial genomes - Mechanism of Gene Transfer - Genome Organization - Plasmids - The features of eukaryotic chromosomes Introduction : - Genetics: the study of genes - Chromosomes: structures containing DNA, the chromosomes contain genes - Genes: segments of DNA that code for functional products, usually proteins - Genome: all the genetic information in a cell Genetic Code: the BIG picture - The genetic code is a set of rules that determines how a nucleotide sequence is converted to an amino acid sequence of a protein Mutation Replication (^) Transcription Translation

Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy ▪ Conjugation Microbial Genetics – DNA

- In 1928, Frederick Griffith discovered transformation in bacteria - In 1944, Oswald Avery and colleagues showed that the transforming substance is DNA - In 1953, Rosalind Franklin used X-ray crystallography to determine that DNA is a double helix - Later In 1953, James Watson, Francis crick and Maurice Wilkins discovered the complementary bases and antiparallel nature of DNA **Mechanisms of Gene Transfer

• DNA-mediated transformation** : the transfer of naked DNA from one bacterium to another - Discovered by Frederick Griffith in 1928 while working with Streptococcus Pneumoniae S strain (heat killed) inject into mice mice live S strain (heat killed)

• inject into mice mice die R strain (live) Griffith Experiment - In the 1950’s, conjugation was discovered o Horizontal gene transfer mechanism requiring cell-to-cell contact, which could transfer large segments of some bacterial chromosomes - Allows genes to be mapped relative to one another according to time of transfer o Results suggested that bacterial chromosomes were circular - We know that there is tremendous diversity in prokaryotic genomes Mechanism of Genetic Transformation in Bacteria

Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy Genome Organization

- prokaryote chromosomes range from 130 to 14,000 kilobase pairs (kb) o Eukaryotic chromosomes can be as big as 100 million kb o Human genome is over 2 million kb - Genomes contain noncoding DNA o > 90 % of eukaryotic genomes o Only < 15 % of prokaryotic genomes - Prokaryotic genomes includes extrachromosomal DNA (plasmids) - Kb = kilobase pairs = 1000 base pairs Functional Units of Genes - A gene is the basic physical and functional unit of heredity - A gene can operate independently of others - It may exist in tandem with other genes in a unit called an operon - Humans have bw 20,000 and 25,000 genes DNA Structure - DNA, deoxyribonucleic acid, is a polymer of nucleotides - Each nucleotide consists of three parts: o 1. Nitrogenous base ▪ Purine: adenine and guanine

Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy

1. Cleaving both strands at one site in the molecule 2) passing an intact part of the molecule bw ends of the cut site and 3) reconnecting the free ends Plasmids

- 2 kinds of extragenomic DNA molecules can interact with bacterial genomes: o Horizontally transferred plasmids o The genomes of bacteriophages (viruses that infect bacterial cells) - Plasmid-encoded functions can contribute to the physiology of the cell o For example, antibiotic resistance Eukaryotic Chromosomes - Eukaryotic genomes are larger than those of bacteria - Because their chromosomes are linear , eukaryotes require a reverse transcriptase called telomerase to replicate their ends - Eukaryotic cells pack their DNA within the nucleus using proteins called histones - A large portion of eukaryotic chromosomes are composed of noncoding DNA: o Introns and pseudogenes Archaeal Genomes - Archaeal genomes combine features of bacteria and eukaryotes o Like bacteria, archaea have: ▪ Polygenic operons ▪ Asexual reproduction ▪ Cells lacking a nuclear membrane ▪ A single circular chromosome o In most species of archaea, however, the processes of DNA replication, transcription, and translation more closely resemble those of eukaryotes - Archaea are not medically important because their optimal living conditions are not similar to human body environment - However, archaea can accept DNA from other organisms which could cause problems in the future

Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy Summary

- A genome is all the genetic info that defines an organism - The prokaryotic genome is typically a single, circular chromosome, whereas the eukaryotic genome consists of multiple, linear chromosomes - The DNA structure consists of a double helix, composed of four different nucleotides - The bacterial chromosome is packed in a series of protein-bound loops collectively called the nucleoid - Topoisomerases are enzymes that supercoil DNA - Plasmids are autonomously replicating, extra-chromosomal DNA elements o They benefit the host under certain conditions Bacterial DNA Replication Chapter Overview - Overview of bacterial DNA replication - Terminating replication - Plasmid replication DNA Replication - Microbial DNA needs to replicate itself as accurately and as quickly as possible - Bacterial replication involves a number of proteins and genes coming together in a complex machine o E. coli have a doubling time of about 20 minutes o But, it takes 40 minutes just to copy its chromosome o E.coli genome = 4.6 or 4600 depending on unit - Multiple DNA replication can happen at the same time Overview of Bacteria DNA Replication - Replication of cellular DNA is most cases is semiconservative o Daughter cell receives one parental and one newly synthesized strand - Replication is bidirectional o Start at a fixed origin and progress in opposite directions

Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy DNA Replication – summary

- DNA polymerase III adds nucleotides to the growing DNA strand - In the 5’ 3’ direction - Initiated by an RNA primer (DNA primase) - Leading strand is synthesized continuously, - Lagging strand is synthesized discontinuously, creating **Okazaki fragments

• RNase H** removes RNA primers; Okazaki fragments are joined by the DNA polymerase I and **DNA ligase Major Proteins involved in DNA replication include:
• DnaA:** initiator protein - DnaB: helicase, unwinds double helix - DNA primase: synthesis of RNA primer

Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy

- DNA Pol III: major replication enzyme - DNA gyrase: relieves DNA supercoiling - DNA Pol I: replaces RNA primer with DNA - DNA ligase: joins Okazaki fragments Energy Replication is supplied by necleotides Terminating Replication - There are as many as ten terminator sequences (ter) on the Escherichia coli chromosome - A protein called Tus (terminus utilization substance) binds to these sequences and acts as a counter-helicase - Ringed catenanes formed at the completion of replication are separated by topoisomerase IV and XerCD proteins Plasmids - Two kinds of extragenomic DNA molecules can interact with bacterial genomes: o Horizontally transferred plasmids o The genomes of bacteriophages (viruses that infect bacterial cells) - Plasmid-encoded functions can contribute to the physiology of the cell o For example, antibiotic resistance Plasmids Replicate Autonomously - Plasmids are much smaller than chromosomes o Found in archaea, bacteria, and eukaryotic microbes

Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy

- How proteins are degraded **RNA Structure

• RNA** , ribonucleic acid, differes from DNA: o Usually single-stranded ▪ RNA-RNA double strand called hairpins o Contains ribose sugar o Uracil replaces thymine Different Classes of RNA - There are several classes of RNA, each designed for a different purpose o Messenger RNA (mRNA): encodes proteins o Ribosomal RNA (rRNA): shuttles part of ribosomes o Transfer RNA (tRNA): shuttles amino acids o Small RNA (sRNA): regulates transcription or translation o tmRNA: frees ribosomes stuck on damaged mRNA o Catalytic RNA: carries out enzymatic reactions - Some RNA act as scaffolds to build/transport or act as an enzyme and facilitate the chemical reaction The Central Dogma - The cell accesses its vast store of data in its genome by: o Reading a DNA template to make an RNA copy (transcription) o Decoding the RNA to assemble protein (translation) - the chromosome is huge compared to RNA so in this process small copies are made of DNA in form of RNA to make multiple copies of protein - every time we unwind double stranded DNA it is exposed to enzymatic reaction - DNA is a master copy while RNA is a zerox copy - Every time that info is transferred form RNA to protein the RNA has to be recycled Functional Units of Genes - A gene is the basic physical and functional unit of heredity - A gene can operate independently of others - It may exist in tandem with other genes in a unit called an operon Transcription: BIG picture

Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy

- Coding strand – same as info on transcribed DNA however T becomes U - Know how to relate RNA to double stranded DNA - Nucleotide can only be added to 3’ end on RNA Transcription - Transcription in the synthesis of a strand of RNA from a DNA template by RNA polymerase (RNA pol) - Begins when RNA pol binds to the promoter on DNA o Promoter is a sequence of DNA upstream of the transcription start site (+) **RNA Polymerases and Sigma Factor

• RNA pol** is a complex enzyme that carries out transcription by making RNA copies (transcripts) of a DNA template strand - in bacteria, the RNA pol is composed of:

Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy ▪ Relies on a NusA protein ▪ Contact bw NusA and RNA pol cause termination o what determines when transcription is going to end? translation Different Classes of RNA

- there are several classes of RNA, each designed for a different purpose: o messenger RNA (mRNA): encodes proteins o ribosomal RNA (rRNA): integral part of ribosomes o transfer RNA (tRNA): shuttles amino acids o small RNA (sRNA): regulation transcripts or translation o tmRNA: frees ribosomes stuck on damaged mRNA o catalytic RNA carries out enzymatic reactions Antibiotics that Affect Transcription - antibiotics must meet two fundamental criteria: o they must kill or retard the growth of a pathogen, and they must not harm the host - Rifamycin B o Selectively binds to the bacterial RNA pol o Inhibits transcription initiation - Actinomycin D o Nonselectively binds to DNA o Inhibits transcription elongation Translation of RNA to protein - Message in mRNA are arranged in a sentence of triplets of nucleotides, called codons , represent individual amino acids - Ribosomes are the machines that read the language of mRNA and translate it into protein o They do so via the genetic code o And tRNA decodor molecules that convert the language of RNA into that of proteins The Genetic Code - There are 64 possible codons: o 61 specify amino acids ▪ Include the start codons o 3 are stop codons (UAA, UAG and UGA) - The code is degenerate or redundant o Multiple codons can encode the same amino acid o E.g. GCU, GCC, GCA, GCG code for **Alanine tRNA

• tRNA** has clover leaf shape - a tRNA molecule has 2 functional regions: o anticodon : hydrogen bonds with the mRNA codon specifying an amino aicd o 3’ (acceptor) end : binds the amino acid

Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy

- tRNAs contain a large number of unusual, modified bases Codon-anticodon pairing - the tRNA anticodon consists of three nucleotides - the anticodon hydrogen bonds with the mRNA codon in an antiparallel fashion - tRNA is “ charged ” with an amino acid covalently attached to the 3’ end - cell has generally 20 of these “match and attach” tRNA, one for each amino acid

Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy ▪ Located 4-8 bases upstream of the start codon in E. coli o This Shine-Dalgarno sequence is complementary to a sequence at the 3’ end of 16S subunit The Three Stages of Protein Synthesis

- Polypeptide synthesis occurs in 3 stages: o 1. Initiation – brings the two ribosomal subunits together, placing the first amino acid in position o 2. Elongation – sequentially adds amino acids as directed by mRNA transcript o 3. Termination – releases the completed protein when it encounters a Stop Codon - Each phase requires a number of protein factors and energy in the form of **GTP Antibiotics that Affect Translation

• Streptomycin** : inhibits 70S ribosome formation - Tetracycline : inhibits translation by binding to 30S subunit - Chloramphenicol and erythromycin : binds to 23S rRNA of the 50S ribosomal subunit Bacterial Transcription and Translation are Coupled - Different ribosomes can bind simultaneously to the start of each cistron within a polycistronic mRNA - Before RNA polymerase has even finished making an mRNA molecule, ribosomes will bind to the 5’ end of the mRNA and begin translating protein o This is called coupled transcription and translation - Eukaryoic microbes, on the other hand, use separate cellular compartments to carry out most of their transcription and translation Post Translational Modification

1. Protein folding
2. Protein degradation
3. Secretion: protein traffic control a. Transmembrane protein b. Protein export to the periplasm Protein Folding - Folding of many proteins requires assistance from other proteins called chaperones o GroEL and GroES chaperones ▪ Form stacked ring with a hollow center ▪ The protein fits inside the open hole o DnaK chaperones ▪ Clamp down on a polypeptide to assist folding - Damaged proteins randomly enter chaperone-based refolding pathways or degradation pathways until the protein is repaired or destroyed

Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy Secretion: protein traffic control

- Proteins destined for the bacterial cell membrane or envelope regions require special export systems - Proteins meant for the cell membrane are tagged with hydrophobic N-terminal signal sequences of 15-30 amino acids o These sequences are bound by the signal recognition particle (SRP) ▪ Proteins then undergo cotranslational export Protein Export to the Periplasm - Many periplasmic proteins, such as SOD and maltose-binding protein, are delivered to the periplasm by a common pathway called the SecA-dependent general secretion pathway Journey through the Outer Membrane - Gram-negative bacteria need to export proteins completely out of the cell o for example, digestive enzymes and toxins - seven elegant secretion systems have evolved: o labeled Type I-VII - Type I moves proteins to the extracellular environment Protein Vs. Eukaryote Summary - Transcription is the conversion of a DNA template into an RNA transcript o Carried out by the complex enzyme RNA polymerase o The sigma factor recognizes the promoter and the core polymerase elongates the RNA strand, until a termination signal is reached o May or may not depend on the Rho protein