RNA: Transcription and Processing Chapter 8

Definitions to learn Exon Intron Splicesome (RNA splicing) Transcription Transcript

RNA Serves as an intermediate between DNA and Proteins Ribose sugar RNA is usually single-stranded Uracil replaces Thymine RNA can catalyze its biological reactions Similar to proteins but unlike DNA Ribozyme (RNA molecule that functions like enzyme) Produced in the Nucleus transported to the cytoplasm

4 ribonucleotides found in RNA Thymine is missing

Review time…. What did Avery, Macleod and McCarty do in their experiment? What are the three chemical components of a nucleotide? DNA with a higher WHAT is more stable? DNA is synthesized in what direction? Spontaneous (though infrequent) change of a nitrogenous base to a slightly distinct form is called what? What is the role of Beta clamp? Where do DNAa proteins bind in prokaryotes? What are the 3 proteins needed to assemble the replisome in eukaryotes? Where do you find the DNA primer?

3 Main Classes of RNA mRNA tRNA rRNA

Other Classes of RNA Small nuclear RNAs (snRNA) MicroRNA (miRNA) Short RNAs found in the nucleus, assist in RNA processing MicroRNA (miRNA) Encode RNA that controls the amount of protein produced in eukaryotic cells Small interfering RNAs (siRNA) Short dsRNAs produced by the cleavage of long dsRNA Piwi-interacting RNAs (piRNAs) RNA transcribed by pi-clusters Helps prevent spread of transposable elements Long noncoding RNAs (lncRNAs or ncRNAs) Non-coding pieces of RNA approximately 200nt in length

Transcription Process of transcribing DNA into RNA RNA is called a transcript DNA is the template Both DNA strands can be used as a template For any ONE gene, only one strand is used Starts at the 3’ end of DNA RNA is transcribed 5’ to 3’ direction

Overview of Transcription

Coding vs. noncoding strand

Concept Question Which of the following is evidence that RNA was a message-carrying intermediary between DNA and protein? A) A hydroxyl group is present on ribose. B) RNA is single stranded and thus cannot be copied by semiconservative replication in a manner similar to DNA. C) RNA structure includes a molecular code, proving that it carries a genetic message. D) RNA is produced in the nucleus (with DNA) and then migrates to the cytosol, the location of protein synthesis.

Concept question If the DNA template 5′-ATGCATGC-3′ were transcribed to RNA, the RNA would read: A) 3′ TACGTACG 5′. B) 5′ AUGCAUGC 3′. C) 5′ UACGUACG 5′. D) 3′ UACGUACG 5′.

Stages of Transcription Initiation Elongation Termination Lets start with prokaryotes first….

Initiation begins with the promoter Downstream Upstream Promoter sequence in ecoli UTR – Untranslated Regions (5’ UTR or 3’ UTR)

RNA polymerase holoenzyme Bacterial RNA polymerase complex that scans DNA for a promoter sequence (sigma factor binds) The RNA pol holoenzyme is a multi-subunit complex Alpha subunits help assemble the enzyme and promote interaction with regulatory proteins, Beta subunit is active in catalysis Beta prime binds to DNA And omega has roles in enzyme assembly and regulation of gene expression Sigma binds to the -10 and -35 regions which positions the holoenzyme to initiate transcription correctly at the start site Sigma also helps separate DNA star around the -10 region so the core enzymes can bind tightly to the DNA in prep for RNA synthesis After core enzyme is bound, transcription begins and the sigma subunit dissociates from the rest of the complex

E. coli σ factors More than one σ factor. σ70 has a mass of 70 kilodaltons Primary σ for initiation of transcription in most E.coli genes Other σ factors recognize different promoter sequences Therefore, associating with different σ factors allows the same core enzymes to recognize different promoter sequences

Other σ factors in bacteria σ19 - the ferric citrate sigma factor, regulates iron transport σ28 - the flagella sigma factor σ24 - the extreme heat stress sigma factor σ32 - the heat shock sigma factor, it is turned on when bacteria are exposed to heat. σ38 - the starvation/stationary phase sigma factor σ54 - the nitrogen-limitation sigma factor

Elongation and Termination Transcription bubble GC rich The energy for the addition of a nucleotide is derived from splitting the high –energy triphosphate and releasing inorganic diphosphate, using this equation 8 to 9 nt make up the RNA-DNA duplex inside the transcription bubble Complementary pairs bonds are broken as RNA strand leaves the bubble Termination occurs after the end of the protein encoding gene segment in the 3’ untranslated region (3’ UTR) Two mechanism for termination in E Coli – instrinsic and rho dependent. Intrinsic the termination is direct- the termination sequences contain about 40 base pairs ending in a GC rich stretch of A which results in the formation of a hair pin loop. The loop cause the RNA pol to fall off Rho dependent- a protein is used to help recognize a nucleotide sequence that is rich is C and low in G which includes un upstream segment called rut. Rho binds to rut causing Polymerase to pause and disociate the RNA for the polymerase RNA Polymerase pauses, it’s blocked, and then falls off

Binding of Rho to rut, RNA Polymerase Pause, and dissociation of RNA Utilization site No string of U’s Binding of Rho to rut, RNA Polymerase Pause, and dissociation of RNA

Concept Check Why does E. coli have several different sigma factors? A) They allow different RNA polymerases to bind to the promoters. B) They allow the different subunits of the RNA polymerase holoenzyme to bind to each other. C) There is no good reason. They all perform the same function. D )They allow RNA polymerase to recognize and bind to a different subset of promoters.

Transcription in Eukaryotes Similar to transcription in prokaryotes but more complicated 3 primary reasons for more complexity Larger eukaryotic genome (more genes) Presence of the nucleus Genomic DNA is organized into chromatin in eukaryotes