RNA structure, synthesis, processing, and transcription

what is the flow of genetic information?

• the central dogma
  → DNA: replication
  → transcription in the nucleus
  → RNA
  → translation in the cytoplasm
  → ribosomes: make proteins

what is the basic structure of RNA?

• nucleotides are ribonucleotides

• made with ribose as the sugar

• nucleotides bound by a phosphodiester bond (phosphate group + sugar + base)

• single stranded

what are the nucleotides of RNA?

adenine, guanine, cytosine, uracil

what is messenger RNA (mRNA)?

codes for proteins

what is the major category of RNA in cells?

rRNA

what is transcription?

• process of transcribing the template strand of DNA into mRNA
• involves H-bond formation between bases on RNA and their complementary bases on DNA
• bonds are later released as the RNA strand separates from the DNA
• produces RNA complementary to one strand of DNA
• transcription can occur quickly, multiple genes can be transcribed at once

what are the significant differences of transcription compared to DNA synthesis?

• the RNA strand does not remain H-bonded to the DNA template strand
• the RNA is displaced and the DNA helix reforms
• RNA molecules are shorter: only a portion of the DNA gets transcribed at any time
• most RNA molecules are less than a few thousand nucleotides long

what are the components of the gene locus?

• 5' end: upstream regulatory elements, enhancers, promoter
• introns and exons: transcription start site (TSS), coding sequence
• 3' end: downstream regulatory elements, enhancers

what are the types of proteins involved in transcription?

RNA polymerase and transcription regulators

what is RNA polymerase?

• enzyme that catalyzes transcription: formation of the phosphodiester bond that links the nucleotides in the chain

• moves along the DNA, unwinding part of the double helix

• new RNA chain then grows, extending in the 5' to 3' direction

• can start transcription without a primer and can unwind the DNA

• may make more mistakes, but is less of a problem because it does not get passed down to future generations

what are the polymerases in eukaryotic cells?

RNA polymerase I, II, III

what is RNA polymerase II?

transcribes mRNA

what is rifadin (rifampicin)?

• antibiotic that inhibits bacterial RNA polymerase
• suppresses transcription of genes in bacteria
• lack of transcription of essential bacteria genes eventually leads to death of the bacteria
• useful for treating infections or diseases caused by infections (e.g. tuberculosis, etc.)

what are the basic steps of transcription?

1. initiation
2. elongation
3. termination
4. processing of RNA

what are the steps of initiation?

1. binding of TFIID to the TATA box
2. binding of the general transcription factors to the promoter
3. binding of transcription regulators to enhancers

what is binding of TFIID to the TATA box?

• binding via its TBP (TATA binding protein: subunit of TFIID)
• first step: TFIID (transcription factor II D) to the gene's promoter
• leads to a change in DNA conformation important for subsequent steps

what is the TATA box?

• a sequence within the promoter that is rich in As and Ts
• ~25-35 bp upstream to the transcription start site (TSS)

what is binding of the general transcription factors to the promoter?

• initiation complex: general transcription factors together with RNA pol II
• after TFIID binds, other proteins bind to this region of DNA along with TFIID, directly or indirectly
• general transcription factors: TFIIB, TFIIA, TFIIF, TFIIE, TFIIH
• key step for transcription initiation of nearly all genes transcribed by RNA pol II (even without a TATA box)

what is binding of transcription regulators to enhancers?

• in addition to the promoter, many genes also contain enhancers
• binding can contribute to the efficient binding of the intitation complex to the promoter
• transcription activators usually interact with a mediator complex

what is a mediator complex?

• mediates an interaction between the activator and the components of the initiator complex
• allows enhancer binding proteins to communicate with RNA pol II and the general transcription factors
• correctly positions TFIIH near the tail of RNA pol II, facilitating the phosphorylation of RNA pol II

what are enhancers?

• type of cis-regulatory element, often located upstream (5') to the promoter
• set up at the initiation stage
• without enhancers, transcription occurs only at a basal level
• can increase the level of transcription
• bind of activating proteins can contribute to the efficient binding of the general transcription factors and RNA pol II to the promoter

what are enhancer binding proteins?

• have roles in recruitment of chromatin-modifying enzymes: include those that modify DNA and histones
• modification of the chromatin facilitates assembly of the transcriptional machinery

what is transcription elongation?

• starts when RNA polymerase moves along the DNA and starts to add the ribonucleotides, one by one
• ribonucleotides are H-bonded to their complementary deoxyribonucleotides
• phosphodiester bond forms between each ribonucleotide
• addition of ribonucleotides starts with the one that is complementary to the nucleotide at the transcription start site (TSS)

what is TFIIH?

• one of the members of the initiation complex
• facilitates the transition from initiation to elongation
• protein kinase

what are elongation factors?

• play a key role in elongation of the RNA chain
• help ensure that the RNA pol II will not dissociate before reaching the end of the gene
• some may also modify histones
• include Elf1, Spt4, Spt45,…

what is the process of elongation?

→ beginning of transcription and release of initiation factors
→ TFIIH uses energy from the hydrolysis of ATP to pull apart the 2 DNA strands at the transcription initiation point
→ TFIID also phosphorylates multiple serines on the C-terminal domain of RNA pol II
→ leads to the release of the general transcription factors, including TFIIH, from the complex
→ UTP, ATP, CTP, GTP (from the cell's nucleotide pool) become incorporated into the RNA chain and are hydrolyzed: 2 phosphates are released
→ the hydrolysis provides energy for the continued synthesis of RNA
→ as transcription proceeds, the CTD on the RNA polymerase becomes further phosphorylated by other kinases
→ the phosphorylation provides docking sites for the next set of proteins: include elongation factors

what is the process of transcription termination?

→ once the RNA pol II reaches the end of the gene's coding region and encounters the 3' noncoding region, transcription will terminate
→ requires the RNA pol II to encounter certain signals
→ different sequences can signal the RNA pol II to stop
→ RNA pol II will terminate after it transcribes through the G-rich or GU-rich region
→ once the RNA pol II has transcribed the poly-A and G/GU-rich signals, certain factors that were bound to the CTD of RNA polymerase will bind the RNA
→ cleavage factors (CPSF, CSTF) cleave RNA downstream to the poly-A signal but upstream of the G/GU-rich sequence
→ mRNA is released from the RNA pol II complex

what is an important termination signal?

polyadenylation (poly-A) signal at the 3' end of the DNA

what is the CPSF?

• cleavage and polyadenylation specificity factor
• interacts with the poly-A signal on the RNA

what is CSTF?

• cleavage stimulation factor
• interacts with the G/GU-rich region

what are certain factors that were bound to the CTD of RNA polymerase?

• CPSF and CSTF
• recruit additional factors, which include cleavage factors

what is the process of processing RNA after termination?

→ after transcription occurs, the mRNA is considered to be pre-mRNA
→ modifications of the pre-mRNA take place: 5' capping (occurs while RNA is still being transcribed), splicing, polyadenylation
→ mature RNA is transported out of the nucleus

what is the 5' gap?

• modified (methylated) guanosine nucleotide called 7-methylguanosine (m7G)
• helps protect the mRNA from degradation
• helps the cell distinguish the RNA made by RNA pol II
• RNA pol I and III do not produce a cap: lack a CTD
• has an important role in the translation of mRNA in the cytosol
• in the nucleus, binds a CBC (cap-binding complex): helps the mRNA to be further processed and exported

what is the process of 5' capping?

→ the unprocessed RNA sequence has 3 phosphate groups are at the 5' end
→ one phosphate group is removed from the 5' end of the RNA: catalyzed by a phosphatase
→ GMP is made by removing 2 phosphate groups from a GTP
→ GMP is added to the 5' end of the RNA: catalyzed by guanyl transferase
→ methyl transferase adds a methyl group to the terminal guanosine

how are more complex cap structures are made?

by adding 1 or 2 additional methyl groups: Cap0, Cap1, Cap2

what is splicing?

• at each side of an intron/exon junction of the mRNA, there is a consensus nucleotide sequence: similar in all genes, and provides information about where the splicing will take place
• performed by spliceosome: complex that consists of small nuclear ribonucleoproteins (SNRPs: U1-U6) and proteins

what are SNRPs?

• specific protein RNA complexes
• bind to the consensus sequences on the RNA
• bring the 2 exons together, forming the lariat

what is the process of splicing?

→ U1 base pairs with the 5' splice junction (GU)
→ BPP and U2AF bind at the branch point (A) and the 3' splice junction (AG)
→ U2 displaces BPP and U2AF
→ triple complex (U4/U6 + U5) binds along with U2
→ a loop forms and U6 will displace U1
→ NTC/NTR protein complex positions the entire complex to bring the branch point close to the 5' splice site
→ now the A (adenine) at the branch point (in the intron) attacks the 5' splicing site, cutting the sugar phosphate backbone of the RNA
→ the end of the intron now binds to the A, forming a lariat structure
→ the splicosome brings together the 2 exons
→ now the 3' OH group of the first exon reacts with the 5' end of the second exon, and the lariat is released and degraded

what is alternative splicing?

• presence of introns and exons allows for the possibility of alternative splicing
• increases the diversity of genes that can be produced
• can lead to different proteins with different functions

what is the poly A tail?

• string of up to 250 or more adenine nucleotides added to the 3' end of the mRNA after transcription is complete
• formed after the RNA is cleaved during the termination process, spliced, and the enzyme poly-A polymerase (PAP) catalyzes the addition of adenosine nucleotides to the 3' end of the sequence

what are poly-A binding proteins (PABPs)?

bind to the tail and help protect and stabilize it as it lengthens to its final size

what are the functions of the poly A tail?

important for mRNA stability and export from the nucleus, translation, regulation of gene expression

what is the export of mRNAs from the nucleus?

• mature mRNA is transported out of the nucleus into the cytoplasm
• unneeded RNA, such as spliced-out introns, remains in the nucleus and is destroyed
• processed mRNAs are guided through nuclear pore complexes (NPCs), where they leave the nucleus and enter the cytoplasm
• because the pore is so small, energy is needed to actively transport the mRNA through the pores
• involves nuclear transport receptors

what is the process of DNA to protein?

→ initation of transcription
→ capping, elongation, splicing
→ cleavage, polyadenylation, termination
→ export of mRNAs from the nucleus
→ mRNA degradation
→ initiation of protein synthesis (translation)
→ completion of protein synthesis and protein folding
→ protein degradation