Test Answers on Micro Bio – Microbiology – Flashcards
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Introduction to gene structure and expression in microbes
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oMicrobe properties (shape, metabolism, virulence) determined by genetics oGenetic information stored in genes
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| Prokaryotic Genomes |
o 1 chromosome (2 rare) o 1 or more plasmid (extrachromasomal DNA) o Held in nucleoid in cytoplasm o Chromosome usually circular molecule o Associated with proteins and RNA that stabilize and regulate transcription |
Escherichia coli |
o Single thin circular molecule of DNA with 4.6 x 106 base pairs o 1600um in length if laid straight (800x cell length) o Packaged into 10% of cell’s volume by helicase which folds it into loops of 50k to 100k bases o Human genome contains 3 x 109 base pairs in 46 DNA molecules § 1.6 meters long if stretched out, but packed into 5um in diameter nucleus |
Plasmids |
· Small
· Self-replicating
· Extra-chromosomal
· Circular
· DNA
· Carried in cytoplasm of come microbes · 1% or less of chromosome · genes for nonessential traits that confer survival in some conditions · Types o Fertility or F factor – contain genes for sex pili and enzymes of conjugation o Resistance or R factor – carry genes that confer antibiotic resistance or cell toxin resistance o Bacteriocin – contains genes for toxins that kill other bacteria o Virulence or V factor – carry genes for virulence factors such as toxins Dissimilation – carry genes for degradative enzymes |
Eukaryotic Genomes
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o 1 or more chromosomes in nucleus o Extranuclear DNA in mitochondria and chloroplasts and rarely plasmids § Nuclear DNA contains 99.995 of information § Most have more than 1 chromosome o Chromosomes of linear DNA and histones § DNA would in 10nm beads around histones – nucleosomes § Nucleosomes packed into 30nm diameter fibers called chromatin fibers § Euchromatin – active and loosely packed chromatin § Heterochromatin – tightly packed chromatin § Mitosis – condensing to tightly packed chromosome |
Extranuclear DNA of eukaryotes
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o Mitochondria and chloroplasts o Circular and resembles prokaryote DNA § Only code for 5% of their needed proteins § Rest coded for by nucleus o Plasmids found in some fungi and protozoa
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| DNA Replication |
· Replication goes both directions from origin o Prokaryotes have one origin usually o Eukaryotes have multiple · Methylation o Prokaryotes methylate adenine and sometimes cytosine o Plants and animals methylate cytosine only o Reasons § Regulate gene expression § Mark initiation site for DNA synthesis § Mark their own DNA Distinguish older from newer to help proofreading |
RNA Molecules |
Eukaryotes use RNA molecules (ribozymes) to process pre-mRNA
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Transcription
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· Synthesis of mRNA · Prokaryotes – nucleoid · Eukaryotes – nucleus but also mitochondria and chloroplasts · Three steps o Initiation § RNA polymerase binds to DNA and moves along until promotor § Subunit of RNA polymerase called sigma factor participates in promotor recognition · Eukaryotes use transcription factors (separate proteins) § Once adhered, unwinds, and begins RNA synthesis 10 bases downstream from promoter o Elongation § Sigma factor released § Polymerase moves 3-5 to synthesize 5-3’ § Terminated when reaches terminator site o Termination Self termination mechanism or termination protein
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Comparison of RNA Polymerase and DNA Polymerase |
o RNA polymerase § No helicase § Slower § Uracil used § Lacks proofreading so it makes more errors § Many RNA polymerases can be on same RNA molecule |
Translation
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o Nucleotide bases dictate protein synthesis o Requires § mRNA § Ribosomes § tRNA § GTP § Protein factors o Codons – 3 bases o 64 codons o Genetic code is redundant o 61 sense codons o 3 stop codons or nonsense codons o Start codon § AUG § Methionine in archaea and eukaryotes § N-formyl-methionine in bacteria, mitochondria and chloroplasts o Same code for almost all organisms |
Prokaryotic mRNA
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o Requires no processing o Carries more than one gene usually |
Eukaryotic mRNA
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o Noncoding introns between coding exons o mRNA first called pre-messenger RNA o Has to be processed to remove introns o Ribozymes § RNA molecules that have enzymatic activity § Remove introns and splice exons back together o Contains only one gene o mRNA transported out nuclear pores |
tRNA |
o RNA molecules with anticodon and amino acid attached o About 75 ribonucleotides o Highly folded with loops and turns, and a 3’ amino acid acceptor site o Anticodon complementary with amino acid codon o 3’ end carries amino acid o Each tRNA has own loading enzyme that loads with ATP and attaches amino acids covalently o Some bind only to 2 nucleotides often and allow the third one to wobble |
| Ribosomes |
· rRNA and proteins · Carry out protein synthesis · Prokaryotes o 70 S o 50 S and 30 S subunits § 50 S comprised of 2 single stranded rRNA molecules (23S and 5 S) and 33 structurally different proteins § 30 S subunit comprised of 1 single stranded rRNA molecule (16S size) and 21 structurally different proteins · Eukaryotes o 80 S o 60 S and 40 S subunits § 60 S comprised of 2 single stranded rRNA molecules (25S and 5 S) and 34 structurally different proteins § 40 S subunit comprised of 1 single stranded rRNA molecule (18S size) and 21 structurally different proteins · Prokaryotic 30 S subunit can bind 3 mRNA codons at a time · Large 50 S subunit holds 2 tRNAs in E, P, and A site · A site accepts charged tRNAs · P site holds tRNA with polypeptide chain attached · E is exit site for dischared tRNAs · Difference in ribosomes allow some antibiotics to be specific Erythromycin binds to 23 S rRNA |
Stages of Translation
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o Initiation o Elongation o Termination o All stages require protein factors o Initiation and elongation requires energy supplied by GTP |
Initiation
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o 30S subunit binds to mRNA at AUG start site in P site o tRNA-fmet binds to AUG using GTP o 50S subunit binds to complex forming ribosome-mRNA initiation complex
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Elongation
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· Cyclic process o Charged tRNA binds to A site o Ribozyme in 50S subunit transfers P amino acid to A amino acid o Shifts down one spot with GTP o E site releases old P site amino acid o A site accepts next charged tRNA · Requires elongation factors and energy from GTP · Enormous energy cost o GTP for each tRNA in A site o GTP for each move o ATP to charge tRNA o GTP to initiate o GTP to terminate Multiple ribosomes can work same mRNA |
Termination |
o Ribosome reaches stop codon o Release factors cause dissociation of complex |
Control of Transcription |
o 75% of genes constituitive – not regulated (essential) o 25% regulated to be expressed only when needed § Regulation happens at transcription of genes to mRNA usually § Regulation conserved energy by preventing synthesis of unneeded proteins |
Operon Model
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· Explains how prokaryotic cells regulate genes · Operon is a segment of DNA that contains o Promoter site where RNA polymerase binds o Operator – adjacent regulatory site o One or more structural genes that code for one or more enzymes or proteins · Separate regulatory gene coding for repressor protein that turns operon off when it binds to operator or on when it doesn’t bind · Inducible operons o Not transcribed unless activated by inducer o See figure 7a-61 o When no inducer (lactose) repressor binds to operator and blocks transcription o When inducer present, it binds to repressor and inactivates it o Operon transcription occurs · Repressible operons o Transcribed continually until deactivated by repressor § Repressor binds to corepressor before activated § Tryptophan model in Figure 7a-63 § When corepressor (tryptophan) absent, repressor is inactive and operon is transcribed When corepressor accumulates, binds to and activates repressor and blocks transcription |
Gene control in cells
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o Inducible operons typically are catabolic pathways § Substrate is the inducer o Repressible operons typically are anabolic pathways § End product corepressor o Other mechanisms § Catabolic enzyme genes also controlled by glucose dependent catabolic repression involving cAMP · Cells metabolize glucose first
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