Microbiology Final Exam Review - December 2023 PDF

Microbiology Final Exam Review Tuesday, December 10 8AM TR Ch 8, Transcription and Translation and Protein Processing Translation: The Process of Converting RNA to Protein Translation Definition: ○ Translation is the decoding of the RNA message to synthesize a protein. Ribosomes:...

Microbiology Final Exam Review Tuesday, December 10 8AM TR Ch 8, Transcription and Translation and Protein Processing Translation: The Process of Converting RNA to Protein Translation Definition: ○ Translation is the decoding of the RNA message to synthesize a protein. Ribosomes: ○ Ribosomes are the molecular machines that read the language of mRNA and convert, or translate, the genetic code into proteins. The Genetic Code Codon: ○ A codon consists of nucleotide triplets on mRNA that represent individual amino acids. ○ There are 64 possible codons: 61 codons specify amino acids. 3 codons are stop codons (UAA, UGA, UAG) that trigger the end of translation. Characteristics: ○ The genetic code is degenerate or redundant, meaning multiple codons can encode the same amino acid. ○ In most cases, synonymous codons differ only in the last base. ○ The genetic code operates universally across species with very few exceptions. RNA Polymerases and Sigma Factors RNA Polymerase: ○ Also known as DNA-dependent RNA polymerase, it is an enzyme complex that carries out transcription by making RNA copies (transcripts) of a DNA template strand. ○ Components in Bacteria: Core Polymerase: Required for the elongation phase. Sigma Factor: Required for the initiation phase. ○ Holoenzyme: The complex of core polymerase and sigma factor. Subunit Structure of RNA Polymerase Core Polymerase: ○ Composed of four different subunits: two alpha (α), one beta (β), one beta-prime (β′), and one omega (ω) which is not required for transcription but plays a role in the assembly and maintenance of the core RNA polymerase. ○ The beta-prime subunit houses the Mg2+-containing catalytic site for RNA synthesis, and sites for rNTP (ribonucleoside triphosphate) substrates, DNA 7  Microbiology Final Exam Review Tuesday, December 10 8AM TR substrates, and RNA products. DNA fits into a cleft formed by the beta and beta-prime subunits. ○ The alpha subunit assembles beta and beta-prime subunits into a functional complex and communicates with regulatory proteins that can bind DNA. Sigma Factor: ○ Different functional areas: sigma 1 through sigma 4 (σ1–σ4), interacting with the alpha (α), beta (β), and beta prime (β′) subunits. ○ A single bacterial species can make several different sigma factors. Each cell has a "housekeeping" sigma factor, which keeps essential genes and pathways operating. In Escherichia coli, it is sigma-70 (a 70-kDa protein). ○ Recruits the core enzyme to a specific DNA sequence (promoter) and recognizes consensus sequences at the –10 and –35 positions relative to the start of the RNA transcript (+1). Transcription of DNA to RNA Process: ○ Occurs in three stages: Initiation: RNA polymerase binds to the promoter, melts open the DNA helix, and catalyzes the placement of the first RNA nucleotide. Elongation: The sequential addition of ribonucleotides to the 3′ OH end of a growing RNA chain. Termination: RNA polymerase detaches from the DNA after the transcript is made. ○ Initiation of Transcription: RNA polymerase holoenzyme forms a loosely bound, closed complex with DNA. The closed complex must become an open complex through the unwinding of one helical turn, after which RNA polymerase becomes tightly bound to DNA and begins transcription. The first ribonucleoside triphosphate (rNTP) of the new RNA chain is usually a purine (A or G). ○ Elongation of Transcription: The sigma factor dissociates after about 9 bases have been joined. RNA polymerase continues to move along the template, synthesizing RNA at approximately 45 bases/sec, with a 17-bp transcription bubble forming due to DNA unwinding ahead of the moving complex. Transcription Termination All bacterial genes use one of two transcription termination signals: ○ Rho-Dependent Termination: Relies on a protein called Rho and a strong pause site at the 3′ end of the gene. It does not require the NusA protein. ○ Rho-Independent Termination: Requires a GC-rich region of RNA, 4–8 consecutive U residues, and the NusA protein. The GC-rich sequence forms a stem-loop structure that 8  Microbiology Final Exam Review Tuesday, December 10 8AM TR contacts RNA polymerase, halting nucleotide addition and causing RNA polymerase to pause. The weakened DNA-RNA duplex at the poly-U-poly-A base pairs releases the transcript and halts transcription. Different Classes of RNA Messenger RNA (mRNA): Encodes proteins. Ribosomal RNA (rRNA): Forms ribosomes. Transfer RNA (tRNA): Shuttles amino acids to the ribosome. Small RNA (sRNA): Regulates the stability or translation of mRNA. tmRNA: Frees ribosomes stuck on damaged mRNA. Catalytic RNA: Carries out enzymatic reactions (also called ribozymes), found associated with proteins. RNA Stability RNA: ○ Short-lived, primed for quick responses to environmental change. ○ Stability is measured in terms of half-life, which varies drastically among different kinds of RNAs. The average half-life for mRNA is 1–3 minutes. RNA Degradosome: ○ The major cellular structure to degrade most RNAs in bacteria, composed of an RNase, an RNA helicase, and two metabolic enzymes. Translation: The Process of Converting RNA to Protein tRNA Molecules Function: ○ tRNAs are adaptor RNAs attached to amino acids. ○ Approximately 80 bases in length. ○ Shaped like a clover leaf (2D) with 3 loops, or a boomerang (3D). Functional Regions: ○ Anticodon: Hydrogen bonds with the mRNA codon specifying an amino acid. ○ 3′ (Acceptor) End: Binds the amino acid. Composition: ○ Contain a large number of unusual, modified bases. Transfer RNA Primary Sequence: ○ The letters D, M, Y, T, and Ψ stand for modified bases found in tRNA. ○ DHU (or D) Loop: Contains dihydrouracil, which occurs only in this loop. ○ TΨC Loop: Consists of thymine, pseudouracil, and cytosine bases, occurring as a triplet. Codon- Pairing 9  Microbiology Final Exam Review Tuesday, December 10 8AM TR PairinAnticodong: ○ Codon-anticodon pairs are aligned in an antiparallel manner. ○ The tRNA anticodon consists of 3 nucleotides at the base of the anticodon loop and hydrogen-bonds with the mRNA codon. ○ tRNA molecules begin with a 5′ G and end with a 3′ CCA, "charged" with an amino acid covalently attached to the 3′ end. Attaching Amino Acids to tRNA Charging Process: ○ Aminoacyl-tRNA Synthetases: Enzymes that charge tRNAs with the proper amino acid. Each cell typically has 20 of these proteins, one for each amino acid. ○ Steps: Activation: Amino acid is activated, forming an aminoacyl-AMP via a reaction with ATP. Transfer: The amino acid is transferred to the hydroxyl residue of the terminal adenosine of the tRNA, releasing AMP. Release: The tRNA synthetase disengages, releasing the charged tRNA. ○ Classes: Class I: Aminoacylates at the 2'-OH of the terminal adenosine. Class II: Aminoacylates at the 3'-OH of the terminal adenosine. Modified Bases in tRNA Modification Process: ○ Unusual bases in tRNA are modified post-transcription by specific enzymes. ○ These modifications provide stability and make the tRNA a poor substrate for RNase. The Ribosome, a Translation Machine Function: ○ The ribosome translates the mRNA code into the amino acid sequences of proteins. Composition: ○ Composed of two subunits containing proteins and rRNA. ○ Prokaryotic Ribosome: Contains 21 ribosomal proteins around one 16S rRNA molecule and 33 proteins around two rRNA molecules (5S and 23S). Finding the Start of Translation Start Codons: ○ Mark where translation starts and set the correct reading frame: AUG (90%), GUG (8.9%), UUG (1%), and CUG (0.1%). Shine-Dalgarno Sequence: 10  Microbiology Final Exam Review Tuesday, December 10 8AM TR ○ A purine-rich sequence (5′-AGGAGGU-3′) located 4–8 bases upstream of the start codon, complementary to a sequence of 16S rRNA, and positions the start codon precisely in the ribosome P site. Ribosome as a "Ribozyme" Function: ○ The ribosome makes peptide bonds and stitches amino acids together using peptidyltransferase, which is part of 23S rRNA of the large ribosomal subunit. Stages of Protein Synthesis Stages: ○ Initiation: Brings the two ribosomal subunits together, placing the first amino acid in position. ○ Elongation: Sequentially adds amino acids as directed by the mRNA transcript. ○ Termination: Releases the completed protein and recycles ribosomal subunits. Requirements: Each phase requires protein factors and energy in the form of GTP. Protein Synthesis: Initiation Requirements: In E. coli, initiation requires three small proteins (IF1, IF2, and IF3) to assemble the 50S-30S-mRNA complex with the initiator tRNAfMet set in the P site. Protein Synthesis: Elongation Elongation Factors: ○ EF-Tu and EF-G recycle sequentially on and off the ribosome, binding to the same area. ○ Steps: A charged tRNA enters the A site. The amino acid is covalently attached to the tRNA-bound amino acid in the P site, adding to the polypeptide chain. The ribosome moves forward by one codon in a 5′-to-3′ direction. Protein Synthesis: Termination Process: ○ After the last peptide bond forms, the mRNA stop codon enters the A site. ○ Release factors (RF1 or RF2) enter and bind, causing ejection of the tRNA in the E site and releasing the completed polypeptide from the tRNA in the P site. Polysomes Structure: 11  Microbiology Final Exam Review Tuesday, December 10 8AM TR ○ A cell structure consisting of multiple ribosomes performing translation on the same mRNA, protecting the mRNA from degradation by RNases and enabling the speedy production of multiple protein copies from a single mRNA molecule. Coupled Transcription and Translation Process: ○ Simultaneous building of both mRNA and proteins, occurring in bacteria and archaea, which lack nuclear membranes. Ribosomes attach at mRNA ribosome-binding sites and start synthesizing protein before transcription of the gene is complete. Spatial Localization Location: ○ Coupled transcription and translation occur near the nucleoid, while translation of fully transcribed mRNA occurs at the cell poles. In rod-shaped cells, most ribosomes are located at the poles. Protein Modification, Folding, and Degradation Post-Translational Modifications: ○ Proteins often require modifications after translation to achieve an appropriate 3D structure or regulate activity. ○ Over 900 different types of post-translational modifications serve various roles, such as stabilizing proteins, localizing them to specific cell regions, or regulating their activity. Types of Protein Modifications N-formyl Methionine: May be removed or modified by methionine aminopeptidase/deformylase to regulate protein degradation. Phosphorylation or Methylation: Changes the activity of signal transduction proteins. Acetylation: Attachment of acetyl groups to regulate activity and help stabilize proteins. Lipidation: Covalent attachment of lipids providing a hydrophobic tail to anchor lipoproteins to membranes. Glycosylation: Attachment of sugars important in biofilm formation, virulence, and colonization. Adenylation: Attachment of adenosine 5′-monophosphate to regulate enzyme activity. Chapter Summary Ribosome Composition: ○ Ribosomes are composed of two subunits, each containing proteins and rRNA. Translation Phases: ○ Initiation: Ribosomal subunits come together. 12  Microbiology Final Exam Review Tuesday, December 10 8AM TR mRNA and the initiator tRNAfMet set in the P site. ○ Elongation: Sequential addition of amino acids as directed by the mRNA transcript. Involves elongation factors EF-Tu and EF-G. ○ Termination: The completed protein is released. Involves release factors (RF1 or RF2). Protein Processing: ○ Proteins may be modified and folded after translation. ○ Modifications can include phosphorylation, methylation, acetylation, lipidation, glycosylation, and adenylation, among others. ○ These modifications serve various roles such as stabilizing proteins, localizing them to specific cell regions, and regulating their activity. 13