Introduction to Molecular Biology 940.105 PDF

Introduction to Molecular Biology 940.105 Summer Term 2024 Bachelor Lebensmittel- und Biotechnologie 217 Reingard Grabherr Department of Biotechnology Transcription Transcription DNA RNA...

Introduction to Molecular Biology 940.105 Summer Term 2024 Bachelor Lebensmittel- und Biotechnologie 217 Reingard Grabherr Department of Biotechnology Transcription Transcription DNA RNA is single stranded Transcription RNA Three types of RNA: Translation messenger RNA Protein transfer RNA ribosomal RNA Enzyme: RNA-Polymerase Transcription https://www.youtube.com/watch?v=vLz2A1cjPH8 Catalyzed by RNA-Polymerase(s) migrates along DNA double helix DNA template strand is scanned in 3‘- 5‘ direction Information is incorporated into RNA single strand complementary to DNA template strand (5‘-3‘ synthesis) Substrate: Ribonucleoside Triphosphates (ATP, CTP, GTP, UTP)  Transcription G A single template strand of DNA double helix is read and transcribed into RNA RNA structurally quite similar to DNA However: C A Ribose instead of 2-deoxyribose Uracil instead of Thymine RNA DNA Single stranded Double stranded AGCU ACGT Ribose Deoxyribose RNA Transcription vs. DNA Replication Initiation of transcription does not depend on RNA primer Synthesis of ribonucleic acid instead of deoxyribonucleic acid Less accurate than DNA replication: 1 error/104 nucleotides RNA-Pol lacks proofreading activities RNA transcripts in general, shorter than DNA replication units RNA is more abundant in a cell, it is more labile, more flexible, more reactive Transcription mRNA Transcription starting point: promoter Transcription end point: transcription terminator Transcription in bacteria mRNA Promoter (red): typically 5‘-region “upstream“ of transcribed region – contains cis-acting recognition sites for RNA Polymerase and transcription regulators Transcribed region (blue): transcriptional start site, protein coding sequence, which is translated into protein Terminator (yellow): contains signals that are required for termination of transcription per definition 3 phases Initiation Elongation Termination  Promoter mRNA Bacteria: A promoter is a DNA sequence onto which the transcription machinery binds and initiates transcription Located upstream of the gene they regulate. The specific sequence of a promoter is very important because it determines whether the corresponding gene is transcribed all the time, some of the time, or infrequently. At the -10 and -35 regions upstream of the initiation site, there are two promoter consensus sequences Transcription in bacteria Transcription Initation: During the first stage, initiation, RNA polymerase binds to the DNA and finds its start sequence. A sigma factor which assists RNA polymerase in reading start signals from the DNA must be present for the initiation stage unwinding, opening The transcription elongation phase begins with the release of the sigma factor from the polymerase. The dissociation of the sigma factor allows the core enzyme to proceed along the DNA template, synthesizing mRNA in the 5′ to 3′ direction at a rate of approximately 40 nucleotides per second. termination is controlled by the rho factor, which tracks along behind the polymerase on the growing mRNA chain. Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls. As a result, the rho protein collides with the polymerase. The interaction with rho releases the mRNA from the transcription bubble. Biology 2e. Provided by: OpenStax. Located at: http://cnx.org/contents/[email protected]. License: CC BY: Attribution. License Terms: Access for free at https://openstax.org/books/biology-2e/pages/1-introduction  Transcription in bacteria Sigma factors are multi-domain subunits of bacterial RNA polymerase (RNAP) that play critical roles in transcription initiation, including the recognition and opening of promoters as well as the initial steps in RNA synthesis. E. coli: Number of amino Consensus binding Factora Gene Size (kDa) Genes regulated acid residues siteb TTGACA–N17– σ70 (σD) rpoD 613 70 Housekeeping TATAAT CTGGCAC–N5– Nitrogen σ54 (σN) rpoN (ntrA) 477 54 TTGCA metabolism TTGACA–N12– σS rpoS (katF) 362 38 Stationary phase TGTGCTATACT CTTGAA–N14– σ32 (σH) rpoH (htpR) 284 32 Heat shock CCCCATNT TAAA–N15– σF (σ28) fliA 239 28 Flagellar proteins GCCGATAA GAACTT–N16– σE rpoE 191 24 Extreme heat shock TCTGA σfecI fecI 173 19 GGAAAT–N17–TC Iron transport R.R. Burgess, in Encyclopedia of Genetics, 2001  Transcriptional Regulation in Prokaryotes: Promoter Promoters in bacteria contain two short DNA sequences located at the -10 (10 bp 5' or upstream) and - 35 positions from the transcription start site (TSS). Their equivalent to the eukaryotic TATA box, the Pribnow box (TATAAT) is located at the -10 position and is essential for transcription initiation. The -35 position, simply titled the -35 element, typically consists of the sequence TTGACA and this element controls the rate of transcription. Bacterial cells contain sigma factors which assist the RNA polymerase in binding to the promoter region. Operons Operons are a cluster of different genes that are controlled by a single promoter and operator. Operons consist of a promoter, which is recognized by the RNA polymerase, an operator, a segment of DNA in which a repressor or activator can bind, and the structural genes that are transcribed together. Negative repressible operons, are normally bound by a repressor protein that prevents transcription. When an inducer molecule binds to the repressor, it changes its conformation, preventing its binding to the operator and thus allowing for transcription. The Lac operon in bacteria is an example of a negatively controlled operon. A positive repressible operon works in the opposite way. The operon is normally transcribed until a repressor/corepressor binds to the operator preventing transcription. Bacterial Operon trp Operon  Lac Operon https://courses.lumenlearning.com/suny-wmopen-biology1/chapter/outcome-prokaryotic-gene-regulation/ Transcription mRNA Transcription starting point: promoter Transcription end point: transcription terminator Transcription Initiation in Eukaryotes RNA Polymerase I is an enzyme that transcribes ribosomal more complex: RNAs. distinct cis/trans regulatory signals for each of the different RNA RNA Polymerase II is an enzyme polymerases that transcribes precursors of General Transcription Factors (GTF) act jointly with RNA-Pol core protein mRNAs. stays complex during transcriptional initiation RNA Polymerase III is an enzyme that transcribes tRNAs. https://microbenotes.com/eukaryotic-transcription/ Transcription Initiation in Eukaryotes TATA-binding protein (TBP); recognizes and interacts with TATAbox (-25 to -30 upstream of transcriptional initiation site) additional regulators (TFIIB, RNA Pol II) associate to constitute Transcription Initiation Complex Protein phosphorylation at RNA Pol II C-terminal domain (‘tail’) influences interaction with regulatory proteins as a prerequisite for transcriptional initiation/elongation Enzymes involved in Eukaryotic Transcription RNA polymerase I (RNA Pol I) transcribes the 28S, 18S, and 5.8S rRNA genes. RNA polymerase II (RNA Pol II) transcribes protein-coding genes, to yield pre-mRNA RNA polymerase III (RNA Pol III transcribes the genes for tRNA, 5S rRNA, U6 snRNA, and the 7S RNA associated with the signal recognition particle (SRP) involved in the translocation of proteins across the endoplasmic reticulum membrane. Conserved Binding Sites, the TATA Box Most promoter sites for RNA polymerase II include a highly conserved sequence located about 25–35 bp upstream (i.e. to the 5 side) of the start site which has the consensus TATA(A/T)A(A/T) and is called the TATA box. Since the start site is denoted as position +1, the TATA box position is said to be located at about position -25. The TATA box sequence resembles the -10 sequence in prokaryotes (TATAAT) except that it is located further upstream. https://microbenotes.com/eukaryotic-transcription/ Elongation phase TFIIH is a helicase, uses ATP to unwind the DNA helix TFIIH phosphorylates RNA polymerase II which causes this enzyme to change its conformation and dissociate from other proteins in the initiation complex. The key phosphorylation occurs on a long C-terminal tail called the C-terminal domain (CTD) of the RNA polymerase II molecule. only RNA polymerase II that has a non-phosphorylated CTD can initiate transcription but only an RNA polymerase II with a phosphorylated CTD can elongate RNA. RNA synthesis occurs in the 5’ → 3’ direc4on with the RNA polymerase The RNA molecule made from a protein-coding gene by RNA polymerase II is called a primary transcript. RNA processing The primary eukaryotic mRNA transcript is much longer and localised into the nucleus, Capping and Tailing Initially at the 5′ end a cap (consisting of 7-methyl guanosine or 7 mG) and a tail of poly A at the 3′ end are added. The cap is a chemically modified molecule of guanosine triphosphate (GTP). CAP AAAAAAAAAAA Splicing The eukaryotic primary mRNAs are made up of two types of segments; non- coding introns and the coding exons. The introns are removed by a process called RNA splicing where ATP is used to cut the RNA, releasing the introns and joining two adjacent exons to produce mature mRNA. CAP AAAAAAAAAAA AAAAAAAAAAA CAP CAP Eukaryotes: the five-prime cap (5′ cap) is a specially altered nucleotide on the 5’ end of precurser mRNAs. Mitochondrial mRNA and chloroplastic mRNA are not capped. consists of a guanine nucleotide connected to mRNA via an unusual 5′ to 5′ triphosphate linkage. This guanosine is methylated on the 7 position directly after capping in vivo by a methyltransferase. It is referred to as a 7- methylguanylate cap, abbreviated m7G. Export from the nucleus Stability Translation RNA processing: https://www.youtube.com/watch?v=DoSRu15VtdM  Splicing A nucleophilic attack occurs when an electron-rich species (the nucleophile) "attacks" an electron-deficient species (the electrophile forming a new bond Splicosome joins exons https://www.youtube.com/watch?v=aVgwr0QpYNE First, the 2'OH of a specific branchpoint nucleotide within the intron, defined during spliceosome assembly, performs a nucleophilic attack on the first nucleotide of the intron at the 5' splice site, forming the lariat intermediate. Second, the 3'OH of the released 5' exon then performs a nucleophilic attack at the first nucleotide following the last nucleotide of the intron at the 3' splice site, thus joining the exons and releasing the intron lariat.  Transcriptional Regulation in Eukaryotes: Promoter A promoter is a region of DNA where transcription of a gene is initiated. Promoters control the binding of RNA polymerase to DNA. RNA polymerase transcribes DNA to mRNA which is ultimately translated into a functional protein. Thus the promoter region controls when and where in the organism your gene of interest is expressed. Promoters are about 100-1000 base pairs long and are upstream (5’) of the gene. The coding strand is the DNA strand that encodes codons and whose sequence corresponds to the mRNA transcript produced. The antisense strand is referred to as the template strand or non-coding strand as this is the strand that is transcribed by the RNA polymerase. DNA sequences called response elements are located within promoter regions, and they provide a stable binding site for RNA polymerase and transcription factors. Transcription factors are proteins which recruit RNA polymerase  Eukaryotic Promoter Eukaryotes require a minimum of seven transcription factors in order for RNA polymerase II to bind to a promoter. Promoters are controlled by various DNA regulatory sequences including enhancers, boundary elements, insulators, and silencers. It is not unusual to have several regulatory elements such as enhancers several kilobases away from the TSS. RNA molecules as regulators Anti-sense RNA changes mRNA expression Regulates protein concentration within inhibits translation a cell, and thus regulates cell inhibits splicing behaviour As RNA is complementary to target RNA forms double stranded RNA, thereby inhibiting translation or splicing „Molecular Biotechnology“ Clark, Pazdernik Spektrum Akademischer Verlag Double-strand RNA RNA-Interference by dsRNA RNA induced silencing complex asRNA binds to ribosomal binding sites and inhibits translation asRNA inhibits splicing  Micro RNA MicroRNA (miRNAs) modulate gene expression during developmental stages of an organsim Degradation of target RNA Inhibition of translation https://www.youtube.com/watch?v=cK-OGB1_ELE  Differences in transcription between Eukaryotes and Prokaryotes Eukaryotes Prokaryotes Promoters are recognized by transcription factors Promoters are recognized by Sigma factors RNA Polymerase binds to transcription factors RNA Polymerase binds to Sigma factors Enhancers influence promoter activities No mRNA processing mRNA processing (splicing, capping, polyA) Organisation in operons Three different RNA polymerases for Inducible promoters mRNA, rRNA and tRNA One RNA polymerases for Ribosomes recognize and bind to CAP structure mRNA, rRNA and tRNA Ribosomes recognize and bind to Shine Dalgharno Sequence (ribosomal binding site, encoded on the genome downstream of the promoter) The genetic code and the principle of translation are the same for pro- and eukaryotes In Eukaryotes: transcription occurs in the nucleus, mRNA is transported to the cytoplasm where translation occurs Most post translational modifications only happen in Eukaryotes Translation We need: tRNA, amino acids, Ribosomal RNA All cells...translate mRNA to Proteins Transfer RNA Codon-Anticodon tRNA Image source: By Boumphreyfr CC BY-SA 3.0, via Wikimedia Commons  The genetic code The degenerate code, also known as the redundancy of the genetic code, refers to the fact that multiple codons, or sets of three nucleotides, can code for the same amino acid during protein synthesis. Different codons can encode for the same amino acid, but no codon can encode for more than one amino acid. The genetic code 1960: Marshall Nirenberg and Heinrich Matthaei The genetic code There are 64 possible codons that can be formed from combinations of the four nucleotides (A, T/U, G, and C), but there are only 20 different amino acids that are used to build proteins. Therefore, multiple codons can code for the same amino acid, and some amino acids are coded for by as many as six different codons. The degeneracy provides some degree of protection against mutations in the DNA sequence, Higher efficiency of protein synthesis, as it allows multiple tRNA molecules with different anticodons to recognize and bind to the same codon on the mRNA strand. Rare and common codons/tRNAs Codon usage varies between species  Ribosomal ribonucleic acid Ribosomal ribonucleic acid (rRNA) is a non-coding RNA which is the primary component of ribosomes rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosomal DNA (rDNA) and then bound to ribosomal proteins to form a small and large ribosome subunits. rRNA is the physical and mechanical factor of the ribosome that forces transfer RNA (tRNA) and messenger RNA (mRNA) to process and tramslate mRNA into proteins Ribosomal subunits, with RNA in orange and yellow and proteins in blue. https://pdb101.rcsb.org/learn/videos/ribosomal-subunits https://pdb101.rcsb.org/motm/10  Ribosomal binding site Shine–Dalgarno sequence In bacteria AGGAGG Prokaryotes: The Shine–Dalgarno (SD) sequence is a ribosomal binding site in bacterial mRNA, generally located around 8 bases upstream of the start codon AUG. The RNA sequence recruits the ribosome to the mRNA to initiate translation by aligning the ribosome with the start codon. The Shine–Dalgarno sequence is common in bacteria. It is also present in some chloroplasts and mitochondrial transcripts. The six-base consensus sequence is AGGAGG; in E. coli, for example, the sequence is AGGAGGU https://www.youtube.com/watch?v=7cn10wayDug  Ribosomal binding site Eukaryotes: CAP dependent mRNA is transported from the nucleus to the cytoplasm  Some questions: What is a sigma factor? What is a CAP? Differences RNA vs DNA? What is the start codon? What is an anticodon? RNA Pol I, II, III? What is a promoter? What is an operon? What is the operator? What is an enhancer? Degenerate genetic code?  Translation Ribosomal RNA the Ribosome Universally conserved secondary structural elements in rRNA among different species During translation of mRNA, rRNA functions to bind both mRNA and tRNA to facilitate the process of translating mRNA's codon sequence into amino acids rRNA initiates the catalysis of protein synthesis when tRNA is sandwiched between the small subunit (SSU) and large subunit (LSU). In the SSU, the mRNA interacts with the anticodons of the tRNA. In the LSU, the amino acid acceptor stem of the tRNA interacts with the LSU rRNA. The ribosome catalyzes ester-amide exchange, transferring the C-terminus of a nascent peptide from a tRNA to the amine of an amino acid. https://slideplayer.com/slide/4908718/ https://www.youtube.com/watch?v=KZBljAM6B1s CAP dependent translation in eukaryotes interaction of the initiation factors, bound to the 5'-end of an 5’ cap, as well as with the 5’ UTR. These proteins bind the small (40S) ribosomal subunit and hold the mRNA in place. elF3 is associated with the 40S ribosomal subunit and plays a role in keeping the large (60S) ribosomal subunit from prematurely binding. eIF3 also interacts with the elF4F complex, which consists of three other initiation factors: elF4A, elIF4E, and elF4G. CAP-independent translation The best-studied example of cap-independent translation initiation in eukaryotes uses the internal ribosomal entry site (IRES). cap-independent translation does not require a 5' cap to initiate scanning from the 5' end of the mRNA until the start codon. The ribosome can localize to the start site by direct binding, initiation factors, and/or ITAFs (IRES trans-acting factors) bypassing the need to scan the entire 5’ UTR. Examples include factors responding to apoptosis and stress-induced responses. Proteasome Degradation of incompletely/wron folded proteins is mediated by an abundant ATP-dependent protease, called the proteasome Converts the entire protein substrate into short peptides Proteins destined for degradation are marked by polyubiquitin chains that are added via a multistep conjugation process The ubiquitin protein itself consists of 76 amino acids https://www.youtube.com/watch?v=-GwI-UrhpEo By Rogerdodd, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=7677277  Posttranslational modifications function structure folding structure degradation function folding stability membrane trafficking function stability stability function function SUMO (Small Ubiquitin-like Modifier) proteins stability function function Understanding Regulation: Transcriptomics Transcriptomics is defined as the study of transcriptome—the complete set of RNA, also known as expression profiling, as it is a study of the expression levels of mRNAs in a given cell population. https://ib.bioninja.com.au/options/untitled/b4-medicine/dna-microarrays.html  Transcriptomics Micro arrays https://ib.bioninja.com.au/options/untitled/b4-medicine/dna-microarrays.html  Fig 3. Summary of DNA microarrays. Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T (2017) Transcriptomics technologies. PLOS Computational Biology 13(5): e1005457. https://doi.org/10.1371/journal.pcbi.1005457 https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1005457 RNA seq The Biology Notes July 24, 2022 by Mohan Gupta  Fig 4. Summary of RNA sequencing. Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T (2017) Transcriptomics technologies. PLOS Computational Biology 13(5): e1005457. https://doi.org/10.1371/journal.pcbi.1005457 https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1005457  Single cell RNA seq Hwang, B., Lee, J.H. & Bang, D. Single-cell RNA sequencing technologies and bioinformatics pipelines. Exp Mol Med 50, 1–14 (2018). https://doi.org/10.1038/s12276-018-0071-8  Fig 6. identification of gene co-expression patterns across different samples. Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T (2017) Transcriptomics technologies. PLOS Computational Biology 13(5): e1005457. https://doi.org/10.1371/journal.pcbi.1005457 https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1005457 A gene (or genetic) regulatory network (GRN) is a collection of molecular regulators that interact with each other and with other substances in the cell to govern the expression levels of mRNA and proteins which, in turn, determine the function of the cell. GRN also play a central role in morphogenesis, the creation of body structures, developmental biology, onset of diseases, E. Coli GRN DOI:10.1186/1471-2105-5-199  Some questions What is a chaperon? What is transriptomics? What is the proteasome? Posttranslational modifications examples?