DNA to RNA

What is transcription?

DNA to RNA 

• DNA doesn’t direct protein synthesis directly, instead it copies into RNA 

• Only uses ONE strand to make different transcripts based on promoter sequence 

What is consensus nucleotide sequence?

Derived by comparing many sequences with the same basic function and tallying up the most common nucleotides found at each position 

• The nucleotide size graph 

Define DNA supercoiling?

Produced by elongation factors 

• Because of the need to unwind the helix  

• For every 10 open nucleotides = 1 supercoil 

• Topoisomerase helps remove tension during transcription 

• May aid in unwinding DNA from nucleosomes in eukaryotes 

Define general transcription factor?

The transcription factors that eukaryotic RNA polymerases 

require for initiate transcription 

• Are proteins  

• Help position RNA polymerase II at promoter  

• The general TF initiate transcription because DNA packaging via nucleosomes present a problem 

• TFIID, B, A, F, E, & H

What does TFIID do?

Recognizes TATA box (& other DNA sequences) near the transcription start point

What does TFIIB do?

Recognizes BRE element in promoters

• accurately positions RNA polymerase at the start site of transcription

What does TFIIA do?

Stabilize TFIID binding

• not required in all promoters

What does TFIIF do?

Stabilizes RNA polymerase interaction with TFIIB

• helps attract TFIIE and TFIIH

What does TFIIE do?

Attracts and regulates TFIIH

What does TFIIH do?

Unwinds DNA at the transcription start point

• phosphorylates Ser5 of CTD of RNA polymerase

• releases RNA polymerase from the promoter

What is an exon?

part of a sequence that is used/transcribed into mRNA or eventually proteins   

• Expressed coding sequences 

• Transcribed into RNA 

• Spliced together to form a functional gene 

• SR proteins (special serine & arginine rich proteins) assemble on exon to call spliceosomes 

What is an intron

Intervening sequences   

• Transcribed into RNA  

• Removed via splicing 

What is a promoter

a sequence of DNA nucleotides that signal the starting point for RNA synthesis 

• All promoters need general TF for RNA pol. II to begin  

What is RNA polymerase (simple definition)

a group of enzymes that form phosphodiester bonds between ribonucleotides  

What is the central dogma

DNA = (DNA synthesis) > DNA = (Transcription) > RNA = (Translation) > Protein  

* sometimes RNA is the final product and NOT protein 

• For structure/regulation 

What are the 3 RNA polymerases

RNA polymerase I (r)= transcribes 5.8S, 18S, and 28S rRNA genes 

RNA polymerase II (m)= Transcribes ALL protein-coding genes & snoRNA, miRNA, siRNA, lncRNA, and most snRNA genes 

• Has the D, B, A, F, E, & H transcription factors (required) 

RNA polymerase III (t)= Transcribes tRNA, 5S rRNA, and some snRNA genes 

What does RNA pol. I transcribe

transcribes 5.8S, 18S, and 28S rRNA genes 

What does RNA pol. II transcribe?

Transcribes ALL protein-coding genes & snoRNA, miRNA, siRNA, lncRNA, and most snRNA genes 

• Has the D, B, A, F, E, & H transcription factors (required) 

What does RNA pol. III transcribe

Transcribes tRNA, 5S rRNA, and some snRNA genes 

What is snRNA

Small nuclear RNAs 

• Function in nuclear processes (splicing of pre-mRNA) 

  • Join exons together (after being spliced)

What is snoRNA

Small nucleolar RNAs 

• Help process and chemically modify rRNAs

What is miRNA

microRNAs (gene expression regulation) 

• Regulate gene expression by blocking translation of specific mRNAs and causing degradation 

What is mRNA

messenger RNAs 

• codes for proteins 

What is rRNA

Ribosomal RNAs (80% of RNAs) 

• Form the basic structure of ribosome and catalyze protein synthesis 

What is tRNA

transfer RNAs 

• Central to protein synthesis as the adaptors between mRNA and amino acids (at APE site in ribosome) 

What is telomerase RNA

Serves as the template for the telomerase enzyme that extends the ends of chromosomes 

What is lncRNA

long noncoding RNAs 

• Some serve as scaffolds (link non-continuous strands) and regulate cell processes (like X-inactivation) 

What is siRNA

Small interfering RNAs 

• Turn off gene expression by directing the degradation of selective mRNAs and helping to establish repressive chromatin structures  

What are the steps of transcription? (General)

1. TFIID binds to TATA box (double stranded state)

   1. binding causes a distortion in the helix (indicating activity is going on)

2. TFIIB binds to the TBP subunit on TFIID (sometimes TFIIA also binds)

3. TFIIF binds to the RNA pol. II to stabilize TFIIB (attracts E and H)

4. TFIIE & TFIIH bind to RNA pol. II and form pre initiation complex 

5. TFIIH uses ATP to open the DNA & phosphorylates RNA pol. II tail

   1. releasing it from the TF & making a conformational change => Can now begin transcription

6. mRNA processing begins after ~20 nucleotides have been transcribed

7. 5’ cap is added to modify the pre-mRNA transcript (before transcription) 

8. RNA Splicing (during transcription)

9. MRNA 3’ poly A tail (after transcription/splicing)  

10. mature mRNA

What is the initiation process of creating mRNA (TFs)

1. TFIID subunit (TBP) binds to DNA double strand at TATA box (Tata Binding Protein = TFIID subunit) 

2. DNA helix distortion occurs (triggered by TFIID binding) signaling activity is going on 

3. More TF gather (F, E, & H) to form the complete transcription initiation complex 

   • TFIIH does the most work  

     • Uses ATP to open DNA (double strands broken apart) 

     • Next, phosphorylates RNA pol. II tail (CTD) -> conformational change that allows it to transcribe 

       • CTD lengthens, allowing more proteins to bind until they “hop off” to the new RNA strand (to begin 5’ capping, RNA splicing, and poly A tail) 

What is the 5’ capping process?

5. 5’ cap is added to modify the pre-mRNA transcript (before transcription) 

   • Cap = methylated guanine 

     • Acts as marker to let cell know what is being made is mRNA 

       • CBC (Cap-Binding Complex) 

     • Essential for translation 

     • Protects mRNA from degradation 

   • Added by 3 enzymes bound to RNA pol. Tail 

What is the RNA splicing process?

1. Spliceosome forms (6snRNPs) (called by SR proteins on exons) 

2. 2-step enzymatic reaction to remove introns 

3. The 2 neighboring exons are joined together 

4. Exon junction complex protein binds where the intron use to be to indicate that the RNA has undergone splicing successfully 

   • Both exons & introns are transcribed into RNA but introns are removed with splicing 

   • Splicing proteins wait at C terminal domain 

   • snRNA join exons together 

OR

1. snRNPs bind to consensus sequences (form spliceosome to sites)

2. (1^st rxn) 5’ splice site binds to branch point (lariat form) (exon 1 released)

3. (2^nd rxn) 3’ OH in exon 1 piece binds to phosphate in 3’ splice site (releasing intron) 

What is the Poly A tail process?

Consensus sequences (AAUAAAA..CA..~30nucl...GU/U rich region) are bound by binding proteins/enzymes that travel on RNA pol. CTD (tail) 

• Poly-A polymerase adds 200 As to 3’ of mRNA transcript 

• Tail length determined by pol-A binding proteins (stabilize tail and count to 200) 

• When finished, RNA pol. releases the template

How is mature mRNA determined?

look at pick

What are the similarities and differences between transcription and replication

Similarities

Differences

DNA helix unwinds DNA at specific point  One strand serves as the template  RNA polymerized 5’ to 3’ (like DNA)  Template dependent polymerization  Ribonucleotides base-pair with template if it’s a good match (followed by covalently linked)

RNA (single stranded) is removed from the DNA strand, close to where transcription begins  As soon as it’s synthesized so a new transcript can be made before the other is finished  RNA is shorter than DNA  RNA polymerases are transcribers (DNA polymerases are NOT)  Form phosphodiester bonds between ribonucleotides  RNA strands made slower than DNA strands  ~20-50 nucleotides/second  NO PRIMER REQUIRED  Accuracy not as critical  RNA pol. doesn't need 3’ hydroxyl base pair to begin  Only uses ONE strand to make transcripts

Define RNA polymerase (general)

Enzyme that catalyzes the synthesis of an RNA molecule on a DNA template from ribonucleoside triphosphate precursors 

• Can't dissociate from strand until finished (DNA pol. Can remove itself for repair mechanism) 

• Only has modest proofreading capabilities  

What is RNA splicing?

introns (and exons) are removed from primary transcript using protein complex (spliceosome) 

• Occurs within the nucleus  

• Can change the meaning of an RNA molecule 

Splicing: 

• Is flexible (alternative splicing) depending on alternative proteins necessity 

• Depends on: 

  • The affinity of splicing machinery (3 mRNA signals (the 2 splice junctions and branch point) 

  • Spliceosome assembly 

  • Exon definition based on enhancer/activator 

What is a rRNA gene

genes that code for rRNA  

• Found in the nucleolus 

What is rRNA

non-coding RNAs that catylize protein synthesis and have ribosomal function 

• Transcribed by RNA pol. I 

• Doesn't have a cap or Poly A tail so can’t leave nucleus (because pol. I doesn’t have a tail to add this) 

• Needs to be combined with other proteins to work 

• 4 types:  

  • Small subunit: 18s  

  • Large subunits: 5.8s, 28s, & 5s (5s made by pol. III) 

What is snRNA

Small nuclear RNAs that join exons after splicing by recognizing splice sequences 

What is a spliceosome

a large (60s) complex made of RNPs (6 snRNAs + their proteins) 

• snRNAs + 7 protein subunits = snRNP (ribonucleotide proteins) 

  • snRNPs form spliceosome core 

• “complex of multiple snRNPs on mRNA” 

What is a TATA box

A sequence of DNA Ts and As located ~30 nucleotides upstream from transcription start site 

• TFIID binds to it  

• NOT the only transcription start sequence 

  EX: BRE, INR, & DPE (all bind to different general TFII) 

What is a terminator?

Signal in bacterial DNA that halts transcription; in eukaryotes, transcription terminates after cleavage and polyadenylation of the newly synthesized RNA 

• where the polymerase halts and releases both the newly made RNA molecule and the DNA template 

What is a transcription unit

Each transcribed segment of DNA 

• Information in just one gene for just one RNA molecule or single protein (or group of mRNAs if there is splicing) 

• Typically carries the information of just one gene, and therefore codes for either a single RNA molecule or a single protein (or group of related proteins if the initial RNA transcript is spliced in more than one way to produce different mRNAs). 

What is a mediator?

allows activators to communicate (make contact) with transcription complexes 

• Important in regulation of gene expression 

• Chromatin and histone remodeling proteins & enzymes are recruited via a mediator 

What is alternative splicing

Different splicing patterns leading to different protein production 

What is the nucleolus

site of rRNA processing and incorporation of rRNAs into ribosome subunits (ribosome factory) 

• Not membrane bound (allows chromosomal DNA to move around) 

• Contains rRNA genes, precursor rRNAs, mature rRNAs, processing enzymes, and partly built ribosomes (ribosomes assembled outside nucleus) 

  • Telomerase assembled & tRNA processed here 

  • Merge RNA + protein to make complete structures 

How is mRNA exported from the nucleus

Mature mRNA is selectively transported from the nucleus into the cytoplasm 

• Proteins help export (particular proteins indicate mRNA processing is incomplete and NOT ready to leave the nucleus) 

• Requires a nuclear transport receptor (the “key”) to leave the nucleus through the nuclear pore 

What is the function of subnuclear structures

Thought to be involved in processing and storage of RNA-processing components (forms “staging area” for assembly of spliceosome parts) 

• Cajal bodies 

• GEMs 

• Interchromatin granule clusters (“speckles”) 

* all of these (subnuclear aggregates) are needed to get appropriate splicing  

What is disorder that relates to subnuclear structures

Spinal muscular atrophy (SMA) = incorrect splicing because of mutated ribonuclear proteins  

• Mutated SMN1 gene