RNA Structure & Transcription

RNA Structure & Transcription

  • RNA Structure and Function
  • Transcription in Prokaryotes and Eukaryotes
  • Processing of mRNA (textbook Chapter 6)
  • SimBio resources for visualizing transcription/translation
  • Midterm prep module on Quercus for tips and practice questions.
  • BIOB11 #5
  • Study Skills Dossier Check-In

Big Questions

  • How does a genotype become a phenotype?
  • How is this regulated to create cells with different proteins/functions?
  • How/Why do prokaryotes and eukaryotes differ?

Central Dogma of Biology Fig 6-1

  • 1-4: Inheritance, genomes, DNA structure, packaging, repair/change, and replication
  • 5: RNA structure and transcription
  • 5-6: RNA processing
  • 6: Reading the genetic code
  • 7: Translation
  • 8: Regulating transcription
  • 9: Regulating RNA processing & translation
  • 10: Gene editing technologies & regenerative medicine
  • 10-12: Post-translation regulation and cell signaling
  • 11-12: Cancer
  • Midterm content
  • BIOB11 concepts are all connected!

RNA & Transcription Learning Objectives

  • Explain how moving from information storage (DNA) to information use (RNA and proteins) includes amplification.
  • Identify differences & similarities between the structures and functions of RNA vs DNA.
  • Explain the general steps in bacterial transcription, including how the template strand & directionality of DNA is selected and mechanisms involved in initiation and termination.
  • Identify the 5’ to 3’ ends of the RNA (and template/coding DNA strands) during transcription and be able to predict RNA sequences from DNA sequences.
  • Explain what a consensus sequence is and how this might be determined by researchers.
  • Identify differences between eukaryotic and prokaryotic transcription including why they can differ with respect to the ratios of promotors to genes to potential types of proteins produced.
  • Explain what alternative splicing is and how it could impact cell function.
  • Describe the major types of RNA in eukaryotes and which RNA polymerase synthesizes each.
  • Describe roles of each of the following in eukaryotic mRNA transcription and RNA processing: TATA box, GTFs (TFIID, TBP, TFIIH), transcription factors, RNA pol II, chromatin remodeling factors and transcription elongation factors, capping enzyme, RNA methyltransferase, CPSF, CstF, Poly (A) Polymerase
  • Identify functions for the RNA polymerase CTD and the role of CTD phosphorylation
  • Draw a primary transcript of pre-mRNA and a processed mRNA and label the introns vs exons, coding regions, 5’ cap, 3’ poly(A) tail, 3’ UTR, and 5’ UTR
  • Identify functions for the 5’ cap and 3’ poly(A)tail and describe how/when they are added

RNA Structure

  • Single-stranded
  • Has ribose instead of deoxyribose
  • Has uracil instead of thymine (base-pairs with adenine)
  • DNA & RNA both have:
    • A sugar/phosphate backbone and nitrogenous bases
    • 5’-3’ directionality within a strand
    • Complementary base-pairing

RNA Secondary and Tertiary Structure

  • Creates folds and loops that can have functions & may be recognized by RNA-binding proteins.

Types/Functions of RNA

  • mRNAs: Messenger RNAs, code for proteins
  • rRNAs: Ribosomal RNAs, form the basic structure of the ribosome and catalyze protein synthesis
  • tRNAs: Transfer RNAs, central to protein synthesis as the adaptors between mRNA and amino acids
  • Telomerase RNA: Serves as the template for the telomerase enzyme that extends the ends of chromosomes
  • snRNAs: Small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-mRNA
  • snoRNAs: Small nucleolar RNAs, help to process and chemically modify rRNAs
  • lncRNAs: Long noncoding RNAs, not all of which appear to have a function; some serve as scaffolds and regulate diverse cell processes, including X-chromosome inactivation
  • miRNAs: MicroRNAs, regulate gene expression by blocking translation of specific mRNAs and causing their degradation
  • siRNAs: Small interfering RNAs, turn off gene expression by directing the degradation of selective mRNAs and helping to establish repressive chromatin structures
  • piRNAs: Piwi-interacting RNAs, bind to piwi proteins and protect the germ line from transposable elements

Types/Functions of RNA (continued)

  • Messenger RNA (mRNA): The intermediate between DNA and protein – used as a template for protein synthesis by ribosomes in cytoplasm
  • Ribosomal RNA (rRNA): Provide structural support in the ribosome and catalyzes the chemical reaction in which amino acids are covalently linked to each other
  • Transfer RNA (tRNA): Required for translation of mRNA information into polypeptide sequence i.e. convert the language of nucleotides to the language of amino acids.
  • rRNAs and tRNAs are long lived and have complex secondary and tertiary structures that play a role in their functions. mRNA have shorter half-lives

Transcription: From DNA to RNA

  • Transcription produces RNA complementary to one strand of DNA
  • Carried out by enzymes called DNA-dependent RNA polymerases
  • Go from information storage (DNA) to information use (RNA and proteins) in the cell
  • Amplification: 1 DNA sequence can be used to make MANY mRNA copies!

Transcription: Which Strand?

  • Direction of transcription is determined by the orientation of the promoter sequence in the DNA at the beginning of each gene

Promoter Orientation

  • Ideal promoter sequence: ACAT_GATTC
  • Reverse complement: CTTAG_TGTA

Promoter Region in Prokaryotes

  • -10 and -35 element
  • -10 and -35 positions are counted backwards from the site at which transcription begins (= “+1” nucleotide)
  • Consensus sequence for E. Coli promoters

Three Main Stages of Transcription

  1. Initiation: RNA polymerase binds to a specific sequence of nucleotides (promoter sequence). Requires transcription factors (proteins).
  2. Elongation: Open up DNA strands temporarily allowing synthesis of complementary RNA. Ribonucleotides are linked together by phosphodiester bonds (5’ to 3’ like DNA synthesis!)
  3. Termination: In prokaryotes, RNA polymerase reaches a specific signal sequence on the DNA that causes an extended pause in synthesis and release of the RNA.

Transcription Initiation

  • Ribonucleoside triphosphates (rNTPs) are the building blocks instead of deoxyribonucleoside triphosphates (dNTPs) from DNA synthesis

Transcription Elongation and Termination

  • Primary transcript
  • If moving on the template 3’ to 5’, RNA synthesis is 5’ to 3’

Transcription Terminology

  • RNA strand is made 5’ to 3’ (reading the template strand of DNA 3’ to 5’)
  • Template, antisense, or noncoding strand: the 3’ to 5’ sequence of the gene used to make the RNA
  • Sense, coding, or nontemplate strand: 5’ to 3’ sequence of the gene; it matches the 5’ to 3’ sequence of the RNA

RNA Polymerase Overview

  • DNA makes a sharp turn within the active site of the enzyme
  • DNA and RNA exit out of separate channels
  • Only a small portion of DNA is single-stranded at a time

Transcription Initiation in Prokaryotes

  • Sigma Factor (σ) increases the RNA pol’s affinity for promoter sequences. Different σ factors can lead to expression of different genes
  • RNA Polymerase (5 subunits) + sigma factor (σ) is called the RNA Polymerase Holoenzyme

Summary of Transcription in Prokaryotes

  1. Holoenzyme assembles and finds promoter
  2. RNA pol opens up the DNA strands
  3. Transcription starts…and often stops (abortive initiation)
  4. Once ~10-12 RNA nucleotides are synthesized, the polymerase releases sigma factor (σ)
  5. Transcript elongation continues
  6. Transcription ends when a termination sequence is reached

Transcription Termination in Prokaryotes

  • Termination sequences stop transcription.
  • Can be “intrinsic” when stem-loop hairpins cause RNA pol to pause
  • Can be protein-mediated by providing a binding sequence for a protein (Ex. A protein called rho factor (ρ) in bacteria)

Transcription in Eukaryotes vs Prokaryotes

  • Eukaryotes: Transcription and RNA processing in nucleus, translation in cytoplasm.
  • Prokaryotes: Transcription and translation are coupled in the cytoplasm.

Organization of Protein Coding Genes

FeatureProkaryotesEukaryotes
Genes per promoterMultipleOne
mRNA typePolycistronicMonocistronic
Transcription/TranslationCoupledUncoupled

Transcription in Eukaryotes vs Prokaryotes

  • Eukaryotic RNA requires processing in the nucleus (5’ cap, splicing, 3’ poly A tail)
  • Export: RNA is made in the nucleus, but mRNA will be translated in the cytoplasm

Eukaryotic RNA Splicing

  • Eukaryotic RNA splicing removes introns
  • Alternative splicing allows 1 gene to be used for production of multiple different sequences of proteins (splice isoforms)
  • RNA sequences + proteins regulate this process

Eukaryotic Alternative Splicing Example

  • α-tropomyosin gene

Drosophila Dscam Gene

  • Dscam gene for a Drosophila membrane adhesion protein has 115 exons and 38,016 different possible proteins!
  • These splicing isoforms allow each neuron to acquire a unique identity

Transcription in Eukaryotes

  • Three major types of RNAs (rRNA, mRNA, tRNA) as well as microRNAs are derived from precursors that are larger than the final RNA product (not true in prokaryotes)
  • Transcriptional unit encoded in the DNA includes promoter, coding sequence and sequences for termination of transcription
  • A primary transcript (or pre-RNA or precursor-mRNA) is the initial RNA molecule synthesized from the template DNA
  • RNA Polymerase II synthesizes all eukaryotic mRNA precursors (pre-mRNA).

RNA Polymerases in Eukaryotes

  • Eukaryotes have three different RNA polymerases, with each being involved in the synthesis of distinct types of RNA

Transcription Initiation in Eukaryotes

  • Transcription in eukaryotes requires general transcription factors (GTFs)
  • Additionally, the pattern / amount of transcription is regulated by presence of other transcription factors (and associated proteins) that bind DNA regulatory sequences (i.e. enhancers)

Consensus Sequences

  • There are different promoter elements & not all are present for all genes.
  • Will focus on the TATA box

General Transcription Factors

| Name | Subunits | Roles in transition initiation |
| :------ | :------- | :-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |------|
| TFIID | 12 | Recognizes TATA box and other DNA sequences near the transcription start point |
| TFIIB | 1 | Recognizes BRE element in promoters; accurately positions RNA polymerase at the start site of transcription |
| TFIIA | 2 | Not required in all promoters; stabilizes binding of TFIID |
| TFIIF | 3 | Stabilizes RNA polymerase interaction with TFIIB; helps attract TFIIE and TFIIH |
| TFIIE | 2 | Attracts and regulates TFIIH |
| TFIIH | 10 | Unwinds DNA at the transcription start point, phosphorylates Ser5 of the RNA polymerase C-terminal domain (CTD); releases RNA polymerase from the promoter |

  • TFIID is composed of TBP and 11 additional subunits called TAFs (TBP-associated factors).

Transcription in Eukaryotes (cont.)

  • TATA box promoter sequence is -24 to -32 from the transcription start site. (-30 in humans). Sequence is either identical or similar to 5’-TATAAA-3’
  • TATA box is the site of assembly of preinitiation complex (PIC) that contains GTFs and RNA polymerase
    1. TFIID recognizes and binds the TATA box through its subunit TBP (TATA binding protein), which binds and bends the DNA
    2. Enables other GTFs (TFIIA and TFIIB) to also bind

Transcription in Eukaryotes (cont.)

  1. RNA polymerase is recruited to the PIC along with TFIIF, followed by TFIIE and TFIIH
  • TFIIH (kinase and helicase) uses ATP hydrolysis to open the DNA double helix at the transcription start point, to expose single-stranded template
  • TFIIH phosphorylates the RNA polymerase II tail (called the C-terminal domain, CTD), which signals that RNA Pol II can begin elongation.
  • Other GTFs are released once elongation begins, but TFIID usually stays bound at the promoter to start another PIC

Transcription Through Nucleosomes

  • Three transcription elongation factors (Spt4, Spt5, and Elf1) help the polymerase transcribe through nucleosomes.

  • These factors can:

    • wedge DNA away from the histone core;
    • destabilize histone–DNA interactions.

  • Also aided by ATP-dependent chromatin remodeling complexes and histone chaperones (that can partially disassemble nucleosomes)

Anatomy of Eukaryotic mRNA

  • The processed mRNA contains a “coding sequence” of nucleotides that will direct the order of amino acids in the encoded protein. Splicing has removed the introns RNA
  • Untranslated regions (5’ UTR or 3’ UTR) are RNA sequences that do not encode protein but play important regulatory roles.
  • 5’ end: methylated guanosine cap
  • 3’ end: 50-250 adenosines → Poly(A) tail

RNA Synthesis and Processing

  • During transcription, proteins involved in RNA processing are carried on the RNA Pol II CTD and transferred to the nascent RNA at the appropriate time
  • Processing can begin as soon as the sequences to be processed have been transcribed.

RNA Synthesis and Processing (cont.)

  • Capping proteins are the first to bind the CTD.
  • RNA molecule is efficiently capped as soon as its 5′ end emerges from the RNA polymerase.
  • CTD sites are phosphorylated switches as RNA pol II continues, which recruits proteins involved in splicing and then 3′-end processing proteins

RNA Pol II CTD

  • Coordinate RNA processing during transcription

Addition of 5’ Cap

  • Methylguanosine capping is initiated immediately after synthesis of 5’ end of RNA
  • Cap prevents 5’ end from being digested by exonucleases
  • Plays a role is transport out of nucleus and initiation of mRNA translation to protein
  • Helps cells to distinguish completed mRNA from other types of RNA molecules.

Addition of 5’ Cap (cont.)

  • Capping enzyme: two active sites for two functions

    1. RNA triphosphatase
    2. Guanylyl transferase

  • Step 1: RNA Triphosphatase removes the terminal phosphate group, leaving a diphosphate

Addition of 5’ Cap (cont.)

  • Step 2: Guanyl transferase adds a GMP in an inverted orientation (5’ end of guanosine faces 5’ end of RNA), which forms 5’-5’ triphosphate bridge

Addition of 5’ Cap (cont.)

  • Step 3: The guanine base is then methylated by RNA methyltransferase.
  • The ribose to which the GMP was attached is also methylated.

mRNA Processing: Addition of 3’ Poly(A) Tail

  • Addition of a string of A nucleotides occurs at the 3’end of the mRNA → polyadenylation
  • In mammalian cells, polyadenylation begins ~20 nucleotides downstream of a special cleavage and poly-A signal sequence (5’-AAUAAA-3’)
  • This sequence acts as a recognition binding site for assembly of the protein complex that carries out polyadenylation
  • Poly(A) tail is variable in length (~200 in humans, ~70-90 in yeast)
  • Protects from digestion by exonucleases
  • Can be used to isolate mRNA from total RNA using oligo dT column (TTTTTTTT… on column base pairs with tail) (significant tool in molecular biology)

mRNA Processing: Consensus Sequences for 3’ Processing

  • CPSF binds here
  • CstF binds here
  • CPSF = cleavage and polyadenylation specificity factor
  • CstF = Cleavage stimulation factor

mRNA Processing: Addition of 3’ Poly(A) Tail (cont.)

  • CstF & CPSF are initially attached to RNA Pol CTD but then get transferred to the RNA
  • Initiates cleavage to create a new 3’ end
  • Poly(A) polymerase can then add adenosines to the new 3’ end, without the need for a DNA template (rare for polymerases!)
  • Poly-A-binding proteins coat the tail

Review Questions

  • Which of the following are TRUE?
    • A) Each gene can be used to make many copies of RNA and each mRNA can be used to make many copies of protein
    • B) RNA molecules are single-stranded and lack complementary base-pairing
    • C) There are different types of RNA
    • D) Repair mechanisms ensure that RNA does not contain mismatched base pairs
    • E) RNA synthesis for all genes in a genome uses the same strand of the double helix as a template
    • F) E. coli transcription terminators contain a -10 and -35 element

Review Questions (cont.)

  • Which of the following are true about making mRNA in eukaryotes?
    • A) mRNA is transcribed by RNA polymerase II
    • B) Translation into protein often begins before synthesis of the RNA transcript is complete
    • C) Transcription terminates when it reaches the TTTTTT… template sequence that encodes the 3’ poly(A)tail
    • D) 5’ capping occurs while transcription of the rest of the pre-mRNA is still ongoing
    • E) The RNA pol II CTD is required for forming the pre-initiation complex, but then is cleaved to allow elongation to begin

General Review Questions

  • Both splice isoforms and an alleles can result in different version of a protein, so what is the difference between them?
  • If the sense strand of a gene sequence is 5’ ACGTA-3’, then what is the RNA sequence produced from that portion of the gene?
  • What is a consensus sequence? Why is it a “consensus” instead of just a single, best sequence?
  • Draw yourself a gene with a transcription bubble that has just started transcription (~ 3 nucleotides base-pairing with the DNA). Label: the 3’ end of the new RNA, where the next nucleotide will be added, the 5’ to 3’ directionality of both DNA strands, and the template and coding strands of the DNA.
  • In the image you drew (or a more detailed one of the chemical structures and interactions between proteins and DNA or RNA), which bonds are covalent and which are not?