1. RNA: Transcription and Processing
Chapter 8

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

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

4. 4 ribonucleotides found in RNA
Thymine is missing

5. 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?

6. Classes of RNA
mRNA
tRNA
rRNA

7. 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

8. 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

9. Overview of Transcription

10. Coding vs. noncoding strand

11. 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.

12. 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′.

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

14. Initiation begins with the promoter
Downstream
Upstream
Promoter sequence in ecoli

15. RNA polymerase holoenzyme
Bacterial RNA polymerase that scans DNA for a promoter sequence
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

16. 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

17. 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

18. Elongation and Termination
Transcription bubble
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

20. 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.

21. 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