RNA and Gene Expression BIO 224 Intro to Molecular and Cell Biology.

RNA and Gene Expression BIO 224 Intro to Molecular and Cell Biology

RNA Molecules Three major classes Ribosomal (rRNA) make up parts of ribosomes Messenger (mRNA) provide RNA copies of genes Transfer (tRNA) smallest of RNAs, bring AAs to site of protein synthesis

Role of RNA DNA does not directly dictate protein synthesis A molecule is needed to take information from DNA to the site of protein synthesis RNA can be made from a DNA template SS molecule, uses ribose as sugar, has pyrimidine U instead of T Characteristics suggested the central dogma of the flow of genetic information in molecular biology DNA RNA Protein mRNA molecules transcribed from DNA, serve as template for translation of proteins

Transcription Similar to DNA replication, but different process Makes a complementary RNA copy of the DNA strand Entire genome never transcribed at once Only transcribe certain genes/gene groups at certain times RNA transcription enzymes need to recognize which gene to transcribe and where to begin RNA polymerase enzyme transcribes RNA from DNA template: mRNA RNA polymerase creates nucleic acid polymer of ribonucleotides Discriminates between DNA and RNA nucleotides Only adds ribonucleotides to polymer, creates only RNA molecule

4.9 Synthesis of RNA from DNA

RNA Polymerase Recognizes start and stop points of DNA molecule Morphologically different from DNA polymerase Both add nucleotides to 3 OH of polymers One recognized in E coli, several identified in eukaryotes Made of subunits, recognizes beginning of gene Binds over region of 60 bp or so, causes DNA to locally unwind for initiation of transcription Promoter is region upstream or at beginning of gene that is bound Specialized short DNA sequence If mutated, cant be bound, doesnt function

7.1 E. coli RNA polymerase

7.5 Structure of bacterial RNA polymerase

Initiation Promoter sequences are upstream and downstream of start site Near promoter is closest to transcription beginning, has conserved sequence: TATAATA (TATA box) Far promoter has conserved sequence: TTGACA RNA subunit allows specific binding to promoter region sequences

7.2 Sequences of E. Coli promoters

Elongation After RNA polymerase binds to promoter DNA is unwound to provide template Unwinds near beginning of gene and provides 3OH for template 2 RNTPs are added for transcription to begin After addition of 10 nucleotides, dissociates and allows continuation of elongation

7.4 Transcription by E. coli RNA polymerase (Part 1)

7.4 Transcription by E. coli RNA polymerase (Part 2)

Termination RNA polymerase recognizes termination signal to end transcription Inverted repeat of GC rich area followed by poly-A tail transcribed to form stem-loop hairpin structure Structure disrupts RNA association with DNA template and terminates transcription Other method involves protein Rho that binds extended SS segments of RNA to cause termination

7.6 Transcription termination

Eukaryote Transcription Similar to that in prokaryotes Have different RNA polymerases divided into classes Class I transcribes rRNAs Class II transcribes mRNAs Class III transcribes tRNAs Mitochondria and chloroplasts have different RNA polymerases

7.11 Structure of yeast RNA polymerase II

Eukaryote Initiation RNA polymerase recognizes promoter sequence Upstream promoter sequence TATAA similar to bacterial TATA box for initiation Also have downstream promoter element; some genes use only this for initiating transcription along with Inr Elongation occurs in similar manner to prokaryotes

7.19 A eukaryotic promoter

7.12 Formation of a polymerase II transcription initiation complex (Part 1)

7.12 Formation of a polymerase II transcription initiation complex (Part 2)

7.13 Model of the polymerase II transcription initiation complex

7.14 RNA polymerase II/Mediator complexes

7.17 Transcription of RNA polymerase III genes

Messenger RNA Vary in size small to very large RNA copy of information in DNA Prokaryote mRNA is translated by ribosomes in the cytoplasm while still being transcribed Prokaryotic mRNAs do not exist for long Often degraded within minutes

Eukaryote mRNA Transcribed as pre-mRNA in nucleus and processed before export Introns removed, 5 end capped with 7- methylguanosine, 3 end polyadenylated with poly-A tail Polyadenylation leads to termination of transcription, important in regulation of translation

7.44 Processing of eukaryotic messenger RNAs

7.45 Formation of the 3 ends of eukaryotic mRNAs

7.47 Splicing of pre-mRNA

7.50 Self-splicing introns

7.52 Alternative splicing in Drosophila sex determination

7.54 Editing of apolipoprotein B mRNA

7.55 Regulation of transferrin receptor mRNA stability

mRNA Translation All mRNAs translated in 5 to 3 direction Polypeptide chains assembled from amino to carboxy terminus Each AA specified by an mRNA codon dictated by genetic code Translation occurs on ribosomes, needs all three types of RNAs plus proteins

Expression of Genetic Information DNA RNA Protein Genes determine protein structure Proteins direct cell metabolism via enzymatic activity Genetic information specified by arrangement of DNA bases Proteins are polymers of 20 AAs determined by sequence that dictates structure and function Mutation in DNA sequence leads to AA sequence change: colinearity

4.8 Colinearity of genes and proteins

The Genetic Code Genetic code allows understanding of how sequence of 4 nucleotides is converted to 20 AAs tRNAs act as adaptor between AAs & mRNAs during translation tRNA anticodons pairs in complementary fashion with mRNA codons for attachment of AA to polypeptide chain Three nucleotides specify each AA 64 codons in genetic code: 61 code for AAs, 3 stop codons Some AAs specified by more than one codon Nearly all organisms use same genetic code

4.11 Genetic evidence for a triplet code

4.12 The triplet UUU encodes phenylalanine

Transfer RNA SS, folded upon themselves into DS section with cloverleaf structure 3 end of tRNA has CCA terminus added after transcription, for AA binding Each AA has specific tRNA Aminoacyl tRNA synthetase enzymes attach AAs to tRNAs in two step process tRNAs bring individual AAs to site of protein synthesis Anticodon loop has three base anticodon sequence specific for particular complementary codon on mRNA tRNA anticodon base pairs with mRNA codon to align AA Redundancy of genetic code allows less stringent base pairing than in other processes Some AAs have more than one codon, more than one tRNA

4.10 Function of transfer RNA

8.1 Structure of tRNAs

7.43 Processing of transfer RNAs (Part 1)

7.43 Processing of transfer RNAs (Part 2)

8.2 Attachment of amino acids to tRNAs

8.3 Nonstandard codon-anticodon base pairing (Part 1)

Protein Synthesis Similar in prokaryotes and eukaryotes Occurs on ribosomes Translation starts at specific sequence near 5 end of mRNA, leaves 5 UTR Eukaryotic mRNAs code single polypeptide chain (monocistronic), prokaryote mRNAs code for multiple polypeptides (polycistronic) Both prokaryote and eukaryote mRNAs have 3 UTRs

8.7 Prokaryotic and eukaryotic mRNAs

Ribosomes Sites of protein synthesis in cells Subunits separated by ultracentrifugation, mass measured in Sphedburg units: 70S in prokaryotes, 80S in eukaryotes Made of rRNA subunits and associated proteins Eukaryote 5S, 5.8S, 18S, 28S rRNAs all transcribed from chromosomes Lower eukaryotes dont produce all four Subunits transcribed as a singe unit by RNA Pol I, to precursor that is processed into 40S and 60S subunits that make up 80S ribosome Prokaryotes have 50S and 30S subunits of 70S ribosome Chloroplast and mitochondrial ribosomes resemble bacterial ribosomes Seen as a series of dots on ER, on nuclear envelope, or in cytoplasm Cells have multiple copies of rRNA genes, actively working cells have most ribosomes

7.15 The ribosomal RNA gene

7.16 Initiation of rDNA transcription

7.42 Processing of ribosomal RNAs

8.4 Ribosome structure (Part 1)

8.5 Structure of 16S rRNA

8.6 Structure of the 50S ribosomal subunit

Initiation of Translation AUG is primary start codon for translation Translation starts with AA methionine in eukaryotes and N- formylmethionine in prokaryotes Signals for initiation codon identification different in prokaryote and eukaryote cells Shine-Dalgarno sequence in prokaryotes precedes mRNA initiation sequence and base pairs with sequence near 3 terminus of 16S rRNA Eukaryote mRNAs bound at 7-methylguanosine cap of 5 terminus before ribosome locates initiation codon Actual initiation of translation not entirely understood

8.8 Signals for translation initiation

Translation Process Divided into stages of initiation, elongation, and termination First step of initiation is binding of initiation factors to the small ribosomal subunit then initiator Met tRNA and mRNA Large ribosomal subunit added to complex for elongation to proceed Eukaryote initiation needs twelve or more proteins, eIFs, to begin Elongation occurs after initiation, to synthesize polypeptide chain

8.9 Overview of translation

8.10 Initiation of translation in bacteria

8.11 Initiation of translation in eukaryotic cells (Part 1)

Translation Process Three sites in ribosome for tRNA binding: P, A, E Initiator tRNA binds to P site, second binds to A site with help of EF and peptide bond forms Translocation moves ribosome three nucleotides along mRNA, putting empty A site over next codon, shifting peptidyl tRNA from A site to P site, and placing uncharged tRNA in E site for release Elongation continues in this manner until stop codon moves into A site Release factors recognize stop codons and terminate translation mRNAs translated by series of ribosomes, sometimes simultaneously

8.12 Elongation stage of translation

8.14 Termination of translation

Regulation of Gene Expression All genes not expressed at all times All genes not expressed in all cells Regulation of gene expression is necessary to ensure production of necessary products in correct cells at most appropriate times Gene expression is regulated at various stages in prokaryotes and eukaryotes, with various methods being used

Transcriptional Regulation Mostly occurs at level of initiation in bacteria Negative regulation: some protein products only transcribed and translated in presence of inducers Conserves unnecessary use of cellular energy to produce RNAs and proteins Multiple genes expressed as operon Operator near transcription initiation site controls transcription Repressor binds to operator to block transcription Inducer must be present to block repressor for transcription to occur Operator is cis-acting, repressor is trans-acting

Transcriptional Regulation Positive transcription control best characterized in E. coli by effect of glucose on genes coding for breakdown of other sugars for alternate sources of energy and carbon If glucose available, enzymes for catabolism of other sugars not expressed If glucose levels drop, CAP binds target sequence 60 bp upstream of transcription start site to initiate transcription of other enzymes

7.7 Metabolism of lactose

7.9 Negative control of the lac operon

7.10 Positive control of the lac operon by glucose

Transcriptional Regulation More complex in eukaryotes Controlled at initiation or elongation Proteins bind regulatory sequences and modulate RNA polymerase activity: repressors, enhancers, promoters Modifications of chromatin structure plays a role Cis-acting sequences regulate eukaryote expression

7.20 The SV40 enhancer

7.21 Action of enhancers (Part 1)

7.21 Action of enhancers (Part 2)

7.22 DNA looping

7.23 The immunoglobulin enhancer

7.32 Action of eukaryotic repressors

7.37 Regulation of transcription by miRNAs

7.39 DNA methylation

7.41 Maintenance of methylation patterns

Translational Regulation mRNA translation regulated in prokaryotes and eukaryotes in response to cell stress, nutrient availability, growth factor stimulation Accomplished by repressor proteins, noncoding microRNAs, controlled polyadenylation, initiation factor modulation mRNA localization used in eggs, embryos, nerve cells and moving fibroblasts, to allow protein production in specific locations at appropriate time

8.15 Polysomes

8.16 Translational regulation of ferritin

8.17 Translational repressor binding to 3 untranslated sequences

8.18 Localization of mRNA in Xenopus oocytes

8.19 Regulation of translation by miRNAs

8.20 Regulation of translation by phosphorylation of eIF2 and eIF2B (Part 1)

8.20 Regulation of translation by phosphorylation of eIF2 and eIF2B (Part 2)

Protein Folding and Processing Polypeptide products must be folded into 3-D conformation to function Some protein products consist of multiple polypeptide complexes Some proteins additionally modified by cleavage or attachment of other functional groups (carbohydrates and lipids)

8.21 Action of chaperones during translation

8.22 Action of chaperones during protein transport

8.23 Sequential actions of chaperones

8.27 Proteolytic processing of insulin

8.28 Linkage of carbohydrate side chains to glycoproteins

8.34 Palmitoylation

Protein Regulation Regulation of enzyme activity necessary for proper catalysis of biological reactions Accomplished through gene expression by regulating protein production, or regulation of protein function and activity in the cell Allosteric binding, phosphorylation, protein degradation used to regulate enzymatic function

8.36 Feedback inhibition

8.38 Protein kinases and phosphatases

8.39 Regulation of glycogen breakdown by protein phosphorylation

8.42 The ubiquitin-proteasome pathway

8.43 Cyclin degradation during the cell cycle

8.44 Autophagy

Disclaimer This workforce solution was funded by a grant awarded under the Presidents Community-Based Job Training Grants as implemented by the U.S. Department of Labors Employment and Training Administration. The solution was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor. The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not limited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership. This solution is copyrighted by the institution that created it. Internal use by an organization and/or personal use by an individual for non-commercial purposes is permissible. All other uses require the prior authorization of the copyright owner.

RNA and Gene Expression BIO 224 Intro to Molecular and Cell Biology.

Documents

Transcript of RNA and Gene Expression BIO 224 Intro to Molecular and Cell Biology.