Online Counseling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik – , India

Introduction Programmes and Courses  SEP–SBI082–CP01-02 SEP–SBI082–Unit 01-02

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.3 Credits  Academic Inputs by Sonali Alkari Counsellor, YCMOU Nagpur Study Centre, Faculty LAD college P.G. D of Biotechnology Research officer Ankur Seeds Pvt Ltd

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.4 How to Use This Resource  Counselor at each study center should use this presentation to deliver lecture of minutes during Face-To-Face counseling.  Discussion about students difficulties or tutorial with assignments should follow the lecture for about minutes.  Handouts (with 6 slides on each A4 size page) of this presentation should be provided to each student.  Each student should discuss on the discussion forum all the terms which could not be understood. This will improve his writing skills and enhance knowledge level about topics, which shall be immensely useful for end exam.  Appear several times, for all the Self-Tests, available for this course.  Student can use handouts for last minutes preparation just before end exam.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.5 Learning Objectives  After studying this module, you should be able to: Define Transcription Explain what is Transcriptional Control? Explain Prokaryotic and Eukaryotic Transcription. Explain different stages of Transcriptional Control State enzymes involved in Transcriptional Control

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.6 Transcription–information Transfer Regulons and Stimulons

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.7 Prokaryotic Transcription  Bacteria have operons– groups of related genes w/ same promoter that are transcribed polycistronically.  Polycistronic RNA – multiple genes transcribed as ONE TRANSCRIPT.  No nucleus, so transcription and translation can occur simultaneously.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.8 Eukaryotic Transcription  No operons – groups of related genes can be on different chromosomes  Each gene has its own RNA transcript (monocistronic)  Transcription and translation separated by nucleus and RNA processing

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.9 Prokaryotic vs. Eukaryotic Transcription:1  Prokaryotic transcription occurs in the cytoplasm alongside translation.  Eukaryotic transcription is primarily localized to the nucleus, where it is separated from the cytoplasm by the nuclear membrane.  The transcript is then transported into the cytoplasm where translation occurs.  Another important difference is that eukaryotic DNA is wound around histones to form nucleosomes and packaged as chromatin.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.10 Prokaryotic vs. Eukaryotic Transcription:2  Chromatin has a strong influence on the accessibility of the DNA to transcriptio factors and the transcriptional machinery including RNA polymerase.  In prokaryotes, mRNA is not modified. Eukaryotic mRNA is modified through RNA splicing, 5' end capping, and the addition of a polyA tail

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.11 Transcriptional Control:3 Transcription Initiation Elongation Termination Processing Capping Splicing Polyadenylation Turnover Translation Protein processing Control of initiation usually most important.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.12 Initiation:1  RNA polymerase simply binds to the DNA and, along with other cofactors, unwinds the DNA to create an initiation bubble so that the RNA polymerase has access to the single-stranded DNA template.  In bacteria, transcription begins with the binding of RNA polymerase to the promoter in DNA.  At the start of initiation, the core enzyme is associated with a sigma factor (number 70) that aids in finding the appropriate -35 and -10 basepairs downstream of promoter sequences.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.13 Initiation:2

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.14 Initiation:3  RNA polymerase  Transcription factors  Promoter DNA RNAP binding sites Operator – repressor binding Other TF binding sites  Sigma factor ( σ) – determines promoter specificity  Core + σ = holoenzyme  Binds promoter sequence  Catalyzes “open complex” and transcription of DNA to RNA: start site of txn is +1 α α ββ’ σ

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.15 Initiation:4 The RNA polymerase is a core enzyme consisting of five subunits:  2 α subunits,  1 β subunit,  1 β' subunit, and  1 ω subunit

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.16 RNAP Binds Specific Promoter Sequences  Sigma factors recognize -10 and -35 sequences  Consensus sequences:-10 and -35

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.17 RNA Polymerase Promoters Deviation from consensus sequence leads to weaker gene expression TTGACAT

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.18 Sigma Factor  A sigma factor (σ factor) is a prokaryotic transcription initiation factor that enables specific binding of RNA polymerase to gene promoters.  Different sigma factors are activated in response to different environmental conditions.  Every molecule of RNA polymerase contains exactly one sigma factor subunit, which in the model bacterium Escherichia coli is one of those listed below.  Sigma factors are distinguished by their characteristic molecular weights. For example, σ 70 refers to the sigma factor with a molecular weight of 70 kDa.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.19 Bacterial Sigma Factors  E.coli has at least eight sigma factors;  The number of sigma factors varies between bacterial species.  Different sigma factors bind RNAP and recognize specific sequences  Helps melt DNA to expose transcriptional start site  Most bacteria have major sigma factor and alternate sigma factors

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.20 Sigma Factors E. coli can choose between 7 sigma factors and about 350 transcription factors to fine tune its transcriptional output An Rev Micro Vol. 57: T. M. Gruber Extreme heat shock/extracytoplasmic

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.21 Structure of Sigma Factor  Sigma factors have four main regions that are generally conserved: N-terminus C-terminus  The regions are further subdivided (e.g. 2 includes 2.1, 2.2, etc.)  Region 1 is found only in "primary sigma factors" (RpoD, RpoS in E.coli).  It is involved in ensuring the sigma factor will only bind the promoter when it is complexed with the RNA polymerase.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.22 Structure of Sigma Factor:1  Region 2.4 recognizes and binds to the -10 promoter site.  Region 4.2 recognizes and binds to the -35 promoter site.  The exception to this organization is in σ 54 - type sigma factors. Proteins homologous to σ 54 /RpoN are functional sigma factors, but they have significantly different primary amino acid sequences.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.23 What Regulates Sigma Factors  Number of copies per cell (σ 70 more than alternate)  Anti-sigma factors (bind/sequester sigmas)  Levels of effector molecules  Transcription factors

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.24 Specialized Sigma Factors  Different sigma factors are activated under different environmental conditions.  These specialized sigma factors bind the promoters of genes appropriate to the environmental conditions, increasing the transcription of those genes.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.25 Bacterial RNAP Numbers  In log-phase E. coli: ~4000 genes ~2000 core RNA polymerase molecules ~2/3 (1300) are active at a time ~1/3 (650) can bind σ subunits. ~1200 σ subunits.  Competition of σ for core determines much of a cell’s protein content.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.26 Elongation:1  One strand of DNA, the template strand (or non- coding strand), is used as a template for RNA synthesis.  As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy.  Although RNA polymerase traverses the template strand from 3' → 5', the coding (non-template) strand is usually used as the reference point, so transcription is said to go from 5' → 3'.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.27 Elongation:2

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.28 Elongation:3  This produces an RNA molecule from 5' → 3', an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar- phosphate backbone).  Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA), so many mRNA molecules can be produced from a single copy of a gene.  This step also involves a proofreading mechanism that can replace incorrectly incorporated bases.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.29 Elongation:4  Prokaryotic elongation starts with the "abortive initiation cycle".  During this cycle RNA Polymerase will synthesize mRNA fragments 2-12 nucleotides long.  This continues to occur until the σ factor rearranges, which results in the transcription elongation complex (which gives a 35 bp moving footprint).  The σ factor is released before 80 nucleotides of mRNA are synthesized

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.30 Termination:1  Bacteria use two different strategies for transcription termination: Rho-independent (more common) Rho-dependent  Rho-independent transcription termination, RNA transcription stops when the newly synthesized RNA molecule forms a G-C rich hairpin loop, followed by a run of U's, which makes it detach from the DNA template.  "Rho-dependent" termination, a protein factor called "Rho" destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.31 Termination:2

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.32 Rho-independent Termination  Termination sequence has 2 features:  series of U residues  GC-rich self-complimenting region  GC-rich sequences bind forming stem-loop  Stem-loop causes RNAP to pause  U residues unstable, permit release of RNA chain

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.33 Rho-dependent Termination  Rho is hexameric protein  base segment of RNA wraps around  Rho has ATPase activity, moves along RNA until site of RNAP, unwinds DNA/RNA hybrid  Termination seems to depend on Rho’s ability to “catch up” to RNAP  No obvious sequence similarities, relatively rare

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.34 What You Learn…  Transcription is information transfer  Transcription control Very important to express genes,repress genes and to prevent wastage of energy.  Initiation,Elongation and Termination are the various stages in transcription  RNA polymerase, Transcription factors, Promoter DNA, Sigma factor are different factors responsible for initiation of transcription.  Different Type of sigma factors  transcription termination can be Rho-independent or Rho-dependent

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.35 Critical Thinking Questions 1.Describe in detail the process of Transcription Initiation. 2.State the role of RNA polymerase and sigma factor in transcrption. 3.State different strategies used for transcription termination in eukaryotes.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.36 Hints For Critical Thinking Question 1.Beginning transcription and machinery required for initation 2.Role and structure of RNA polymerase and sigma factor. 3.What is termination of Transcription control and difference Rho-independent or Rho-dependent termination. © 2007, YCMOU. All Rights Reserved.36

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.37 Study Tips:1  Book1 Title: Genes IX. Author: Benjamin Lewin Publisher: Jones & Bartlett Publishers  Book2 Title: Genetics from Genes to Genomes Author: L. H. Hartwell, L. Hood, M. L. Goldberg Publisher: Tata McGraw-Hill

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.38 Study Tips:2  Book3 Title: Genetics Author: Monore W.Strickberger Publisher: Prentice Hall of India  Book4 Title: Genetics Author:C.Serin Publisher: Tata McGraw-Hill

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.39 Study Tips Microsoft Encarta Encyclopedia Wikipedia the free encyclopedia

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