MCBL121_23W_lecture+5_transcription_translation.pdf

BIOL/MCBL 121 INTRODUCTORY MICROBIOLOGY Lecture 5 Transcription and Translation Copyright Ansel Hsiao 2021 Objectives • Describe the functions of RNA polymerase and sigma factors • Describe the general role of transcriptional regulators • Describe open reading frames • Contrast Rho-dependent and...

BIOL/MCBL 121 INTRODUCTORY MICROBIOLOGY Lecture 5 Transcription and Translation Copyright Ansel Hsiao 2021 Objectives • Describe the functions of RNA polymerase and sigma factors • Describe the general role of transcriptional regulators • Describe open reading frames • Contrast Rho-dependent and Rho-independent transcriptional termination • Contrast eukaryotic vs prokaryotic translation • Explain the role of the Shine-Dalgarno sequence in translation Copyright Ansel Hsiao 2021 Central Dogma • Genes (DNA) -> transcript (RNA) -> protein (function) DNA RNA Copyright Ansel Hsiao 2021 Protein Transcription • First step of gene expression • Copying of DNA segment into RNA • Transcription is guided by un-transcribed DNA regulatory sequence upstream (5’) of the gene -> promoter • Near coding region of gene, vs eukaryotes, where it could be kilobases upstream Copyright Ansel Hsiao 2021 Gene coding region Copyright Ansel Hsiao 2021 Pribnow Box • Yellow= conserved • Pink=variable Copyright Ansel Hsiao 2021 RNA Polymerase • Multi-subunit protein complex that uses nucleic acid template to generate RNA polymer • Core Enzyme: RNA synthesis • Sigma factor (σ): • Only needed for initiation of RNA synthesis (not elongation) • Recognizes promoters by binding to -10 (Pribnow Box) and -35 regions of genes • Guide the core enzyme to initiate transcription • Holoenzyme: core enzyme plus Sigma factor Copyright Ansel Hsiao 2021 Regulating Gene Expression • Cells use different mechanisms to sense and respond to conditions within or outside the cell by changing the expression of genes • Regulatory proteins help a cell sense environmental/internal changes and alter its gene expression to match Copyright Ansel Hsiao 2021 Regulators • Regulatory proteins come in two forms: • Repressors: Bind to regulatory sequences in the DNA and prevent transcription of target genes • Activators: Bind to regulatory sequences in the DNA and stimulate transcription of target genes • Some regulatory proteins must first bind small ligands to be active • The transcription of transcriptional regulators can be regulated as well! Copyright Ansel Hsiao 2021 Molecular Regulation • Many different proteins are needed to handle different aspects • “housekeeping proteins” • Generally required for growth or maintenance of cellular functions • Other proteins are only needed under a limited set of conditions (i.e. in response to stress, or to take advantage of specific nutrients) Copyright Ansel Hsiao 2021 Sigma Factors • Multiple σ factors in one cell to turn on the transcription of different sets of genes responding to different conditions: • σ70 can be seen as the “housekeeping” sigma factor • Alternative sigma factors Copyright Ansel Hsiao 2021 Initiation of Transcription • Sigma factor binds DNA • Sigma factor recruits the core enzyme and scans for the promoter region • Core enzyme unwinds DNA at promoter (Open complex) • Sigma factor is released Copyright Ansel Hsiao 2021 Transcription Elongation • Core polymerase synthesizes RNA strand 5’-3’ • Added base is complementary to the template strand • mRNA has the same sequence as the non-template strand – U instead of T Template strand Non-template strand Copyright Ansel Hsiao 2021 Transcription Termination • Rho-dependent termination • Rho-independent termination Copyright Ansel Hsiao 2021 Rho-dependent termination • Rho is a helicase protein (unwinds nucleic acids) • Binds mRNA and moves along the transcript • When Rho reaches RNAP/RNA/DNA complex, it unwinds the RNA/DNA duplex, and causes RNAP to fall off of DNA Copyright Ansel Hsiao 2021 Rho-independent termination • Polymerase slows at pause site • GC-rich sequence (strong basepairing) forms stem loop (RNA secondary structure) • Stem loop causes RNA polymerase to pause Copyright Ansel Hsiao 2021 Rho-independent termination • Pause site followed by poly U site • DNA-RNA UA base pairs are least stable (even less stable if polymerase is stalled) • mRNA breaks off of DNA polymerase released Copyright Ansel Hsiao 2021 Types of RNA • mRNA—messenger • rRNA—ribosomal (structural, non-coding) • tRNA—transfer • Used to carry information from DNA to protein • Other RNAs regulate transcription • sRNA—small RNAs • regulate stability or translation of specific mRNAs into proteins Copyright Ansel Hsiao 2021 Translation • The decoding of RNA into protein Copyright Ansel Hsiao 2021 Gene coding region Transcriptional start is NOT the same as translational start (start codon) Copyright Ansel Hsiao 2021 Open Reading Frame (ORF) • Orientation of consecutive, nonoverlapping triplets of nucleotides that represent amino acids/stop signals in translation • Triplets of nucleotides – codons • 61 codons -> 20 amino acids (degenerate; multiple codons can encode the same amino acid) • 3 stop codons Copyright Ansel Hsiao 2021 Open Reading Frame (ORF) • Contained within mRNA and located between the translation start codon (AUG) and stop codon • Each transcript has three possible reading frames • Stop codon in same frame as start codon Copyright Ansel Hsiao 2021 tRNA • tRNAs bind individual amino acids • tRNAs have specific shape – cloverleaf structure • tRNAs have 3-base anticodon • Base pair to codons in mRNA • Aminoacyl-tRNA transferases- attach aa to tRNA (charge tRNA ) Copyright Ansel Hsiao 2021 Ribosome • Very large rRNA-protein complexes • 2 subunits (30s and 50s), 52 proteins, 3 rRNAs • 30s subunit – 16s rRNA • 50s subunit – 5s and 23s rRNA • rRNA forms the catalytic center of the ribosome- peptidyltransferase activity • Can bind 1 mRNA + 3 tRNAs Ribosomal large (50s) subunit Copyright Ansel Hsiao 2021 Proteins rRNA Translation • The decoding of RNA into protein • Ribosome-binding site (RBS aka Shine-Dalgarno sequence) on mRNA allows binding to 30s subunit • Shine-Dalgarno site is complementary to sequence at the 3’ end of the 16sRNA Copyright Ansel Hsiao 2021 Prokaryotic Translation • Faster than eukaryotic process • Different ribosome structure • Different initiation and release factors to begin and end translation • Less stable mRNAs compared to eukaryotes • Coupled with transcription Copyright Ansel Hsiao 2021 Translation • Transcription and translation are coupled processes in prokaryotes – no nucleus to separate processes • Ribosomes bind mRNA while mRNA is still being synthesized • Multiple ribosomes bind to each mRNA – proteins are made rapidly Copyright Ansel Hsiao 2021 Copyright Ansel Hsiao 2021 Complementary sequence in 16s rRNA of 30s subunit Translation • Multiple ribosomes bind to each mRNA molecule - polysomes Copyright Ansel Hsiao 2021