BMS 532 RNA Structure and Transcription PDF

RNA Structure and Transcription BMS 532 BLOCK 3 L EC TURE 4 (OV ERA LL LEC T U RE 19)  Objectives 1. Compare and contrast structure and sequence of DNA and RNA and compare and contrast eukaryotic and prokaryotic mRNA 2. Explain how RNA forms higher order structures and identi...

RNA Structure and Transcription BMS 532 BLOCK 3 L EC TURE 4 (OV ERA LL LEC T U RE 19)  Objectives 1. Compare and contrast structure and sequence of DNA and RNA and compare and contrast eukaryotic and prokaryotic mRNA 2. Explain how RNA forms higher order structures and identify sequences that would be capable of forming these structures 3. Summarize the types of RNA discussed and their category of corresponding functions if known 4. Explain what is meant by polycistronic mRNA and how it relates to cellular activity 5. Explain the role of promotors and their importance to gene transcription (additional regulatory elements will be addressed in a later section) 6. Summarize the process of Transcription (with an emphasis on the broad process steps of Initiation, Elongation, and Termination) ◦ Explain what is meant by sense and antisense strands (coding and template strands) 7. Explain the mechanics of Transcription initiation and termination and Compare and contrast prokaryotic and eukaryotic transcription initiation and termination ◦ Explain the overall role of transcription factors, promoters, and termination sequences in the process of Transcription 8. Identify the activity of the different RNA polymerases and how termination of their activity varies 9. Explain the variation in RNAPolII termination and identify the impact for changes in factors impacting RNAPolII termination 10. Assess the consequence for changes in DNA or RNA sequence, mRNA structure, RNA polymerase activity, or transcription factor activity on the process of transcription and the final products generated* *This aim will be continued in future lectures  Introduction to Gene Expression Principles Gene expression involves transcription if the gene only encodes RNA (i.e. long non-coding RNA) Gene expression involves transcription and translation if the gene encodes a protein The structure of the final molecule is determined by the sequence coded by genes The primary structure of an RNA molecule is the sequence of bases complementary to the code supplied in the DNA The primary structure of protein is the amino acid sequence Primary structures define higher order structures such as folding and association with other molecules Overall Process; LO1, LO2  RNA Structure Ribose sugar backbone Synthesis 5’ to 3’ and dependent on 3’OH group Mostly single-stranded though ds variants and dsRNA viruses exist Takes on many complex higher order structures based on hydrogen bonding in complementary base pairing ◦ The same molecule may be capable of multiple conformations but some are restricted in the shapes they generate ◦ 3D shape is essential to function LO1, LO2 Types of RNA Encoding RNA ◦ Messenger RNA (mRNA) Noncoding RNA ◦ Ribosomal RNA (rRNA) ◦ Transfer RNA (tRNA) ◦ Small nuclear RNA (snRNA) ◦ Small nucleolar RNA (snoRNA) ◦ Telomerase RNA (TERC) ◦ MicroRNA (miRNA) ◦ Small interfering RNA (siRNA) ◦ Enhancer RNA (eRNA) ◦ Long non-coding RNAs (lncRNA) ◦ Circular RNA (circRNA) LO1, LO2, LO3  Generation of Small Noncoding RNAs and Their Function (Neurodegenerative Diseases) LO1, LO2, LO3 Watson et al 2019  mRNA Structure and Design mRNA = protein coding RNA transcripts All eukaryotic mRNA is monocistronic ◦ Encodes a single protein Prokaryotic and chloroplast mRNA can be polycistronic ◦ Encode multiple proteins ◦ Leader sequence before first gene and intercistronic regions between genes ◦ Proteins encoded by one mRNA tend to function in the same biochemical or metabolic pathway LO1, LO4 Transcription  Transcription Transcription = production of RNA product complementary to the base sequence of specific genes The RNA product differs from DNA ◦ Typically single-stranded ◦ Contains uracil in place of thymine ◦ Represents only a fragment of the DNA as opposed to an entire DNA strand LO1, LO6  Transcription The nucleotide sequence in the transcribed mRNA is complementary to the base sequence in DNA RNA is copied from the template strand in the 5’-to-3’ direction RNA synthesis does not require a primer and proceeds by the addition of nucleotides to form mRNA chain ◦ RNA polymerase lands at sequences of dsDNA = promoter LO5, LO6  Transcription Summary Initiation, Elongation, Termination Use of specific factors confers each stage Phosphorylation is a key component ◦ Plays a role in the processing of mRNA LO5, LO6, LO7  Overall Process 1. Machinery landing at appropriate location 2. Opening of DNA 3. Utilization of DNA as a template for synthesis of a complementary strand of RNA 4. Release of the growing strand 5. Dissociation of the transcription machinery 6. Reannealing of DNA LO6, LO7  Important Sequences within DNA Promoter ◦ Sequence of DNA recognized by RNA polymerase (5’ to 3’ synthesis) ◦ Transcription Start Site ◦ Indicates which DNA strand will be transcribed (TEMPLATE) Termination Site ◦ Sequence of DNA that signals the end of transcription (End of the Gene) ◦ RNA transcript is released when RNA Transcription factors act to help get the RNA polymerase falls off polymerase to the promoter! LO5, LO6, LO7  Prokaryotic RNA Synthesis: PROMOTERS Promoter = nucleotide sequence IN THE DNA upstream to the transcription start site which is the initial binding site of RNA polymerase and transcription initiation factors Promoter recognition by RNA polymerase is a prerequisite for transcription initiation Many promoters contain a similar DNA sequence ◦ TATAAT = “TATA” box (-10) is a consensus sequence of many promoters Consensus promoter sequence at -35 = TTGACA LO5  Eukaryotic RNA Synthesis: PROMOTERS Promoter sequences in eukaryotes are generally much longer and more complex than those in prokaryotes. LO5  Transcription Initiation PROKARYOTIC EXAMPLE EUKARYOTIC EXAMPLE LO7  Eukaryotic RNA Synthesis: Multiple Polymerases Eukaryotes have several types of RNA polymerases Each polymerase has specific functions in transcription: RNA Pol I ◦ Transcribes ribosomal RNA (rRNA) genes RNA Pol II ◦ Transcribes all protein-coding genes as well as the genes for small nuclear RNAs RNA Pol III ◦ Transcribes other functional RNA genes including tRNA and the 5S component of rRNA LO8 17 LO6, LO7  Template Coding LO6  Termination of Transcription Termination in Eukaryotes has been shown to be polymerase-specific ◦ RNA Pol I: TTFI termination factor ◦ RNA Pol II: can continue to transcribe past the termination sequence ◦ Anti-termination factors keep polymerase associated with DNA and continue transcription ◦ Transcript is cleaved and released prior to dissociation of polymerase; role for 5’ exonuclease (i.e. Xrn2) ◦ Carboxy-terminal domain (CTD) in the polymerase (largest subunit of the polymerase) ◦ RNA Pol III: not as fully understood but evidence of termination sequences LO7, LO8  Termination of Transcription More on RNA Pol II Termination LO7, LO8, LO9 Porrua and Libri 2015  More on Transcription Termination LO7, LO9 Porrua and Libri 2015  Multiple Factors Influence RNAPII Termination Multiple factors influence RNAPol II termination Cellular cues contribute to regulation of termination ◦ Impairment of termination under times of stress Overall, termination is one of the least studied areas still in need of increased explanation and understanding LO9  Impact of Changes in Sequence DNA sequences contribute to both transcription and product sequence Change in the promoter sequence or availability can reduce or increase transcription activity ◦ Changes in sequence that increase RNA polymerase affinity for the promoter would increase transcription ◦ Changes in sequence that decrease RNA polymerase affinity for the promoter would decrease transcription If termination sequences are required, changes in sequence could alter termination of transcription producing products of inappropriate length LO10 Questions What are the main differences between DNA and RNA? (LO1) ◦ Which of the following explains the difference in the sugar backbone of DNA and RNA molecules? How does RNA form higher order structures? (LO2) What are the structural noncoding RNAs? What are the regulatory noncoding RNAs? (LO3) What is the consequence for changes in RNA sequence on higher order structures? (LO2, LO10) Why does a polycistronic mRNA make sense for prokaryotes but not eukaryotes? (LO4) Quick Summaries A FEW SLIDES FOR SUMMARY COMPARISONS Overall Objectives: Connections Between Lectures 1. Assess the consequence for changes in DNA or RNA sequence the processes of replication, transcription, and translation and the function of the final products generated* 2. Compare and contrast Replication, Transcription, and Translation* 3. Explain the functional consequence of changes in DNA sequence on RNA and Protein in terms of structure and function* 4. Identify the cause of a given phenotype/change in functional product* *These aims connect the molecular genetics processes and apply to multiple lectures DNA vs. RNA DNA RNA ◦ Deoxyribose ◦ Ribose ◦ Double-stranded (can be ◦ Typically Single-Stranded single-stranded) ◦ A-U; G-C (Uracil) ◦ A-T; G-C ◦ Primer to initiate DNA synthesis at origins of replication Replication vs Transcription REPLICATION TRANSCRIPTION Restricted in life cycle to preparation in G1 and Occurs throughout cell life/cycle active in S phase Only select regions are copied (small sections Entire Molecule is Copied of DNA used to form small RNA molecules) Proofreading to ensure integrity of copied Additional processing of the material occurs material prior to generating a protein Initiation, Elongation, Termination Initiation Elongation Termination Replication Transcription Translation