RNA Structure and Function

• RNA contains ribose sugar, unlike DNA's deoxyribose, with RNA having an OH group on the 2' carbon and DNA having an H.
• RNA is typically single-stranded, while DNA is double-stranded.
• Single-stranded RNA's structural flexibility allows it to form complex 3D shapes, determined by intramolecular base pairing.
• Sugar-phosphate bonds in RNA are formed at the 5' and 3' positions of the ribose sugar.
• RNA uses uracil (U) in place of thymine (T), and uracil pairs with adenine (A) using two hydrogen bonds.
• Uracil can pair with guanine (G) during RNA folding, but not typically during transcription.
• Ribozymes are RNA molecules that can catalyze biological reactions, similar to protein enzymes.

mRNA (messenger RNA)

• Encodes the information needed to synthesize polypeptide chains (proteins).

Gene Expression

• A gene's DNA sequence is transcribed into RNA; for protein-coding genes, this RNA is translated into a polypeptide.

Cistron

• A segment of DNA or RNA that corresponds to a single polypeptide, including the start and stop codons.

Leader Sequence

• Located at the 5' end of mRNA, transcribed but not translated, can be hundreds of base pairs long.
• Can contain regulatory sequences ("attenuators") that influence the level of gene expression.

Nucleases

• Enzymes that cleave the phosphodiester bonds linking nucleotides in nucleic acids.

Functional RNA

• Can suppress gene expression at various levels and maintain genome stability.
• RNA itself is the final functional product, unlike mRNA which is translated into a protein.

Ribosomal RNA (rRNA)

• Found in all species and functions in translation.
• Major components of ribosomes, guiding the assembly of the amino acid chain by mRNAs and tRNAs.

Transfer RNA (tRNA)

• Found in all species and functions in translation.
• Responsible for bringing the correct amino acid to the mRNA during translation.
• rRNA and tRNA work together to facilitate protein synthesis.

Small Nuclear RNA (snRNA)

• Found in eukaryotes and functions in splicing and mRNA processing.
• snRNAs further process RNA transcripts in eukaryotic cells; together with proteins, they form the spliceosome to remove introns from RNAs.
• snRNPs (small nuclear ribonucleoproteins) combine with pre-mRNA and other proteins to form the spliceosome.

MicroRNA (miRNA)

• Found in eukaryotes and functions in translational regulation.
• miRNAs regulate the amount of protein produced by eukaryotic genes.

Small Interfering RNA (siRNA)

• Found in eukaryotes and functions in protection against viral infection.
• Double-stranded RNA molecules that can inhibit virus production through the RNA interference pathway.

Piwi-Interacting RNA (piRNA)

• Found in eukaryotes and functions in genome stabilization.
• piRNAs maintain genome integrity through gene regulation and by preventing the spread of transposable elements.

Long Noncoding RNA (lncRNA)

• Found in eukaryotes and functions in transcription, splicing, and transport regulation.
• lncRNAs may be involved in dosage compensation and chromatin modification (epigenetic regulation).

Steps in Transcription

• Synthesis of RNA from a DNA template.
• The two DNA strands separate, and one strand acts as a template for RNA synthesis.
• RNA synthesis starts at the 3' end of the DNA template and proceeds in the 5' to 3' direction.
• Ribonucleotides form pairs with complementary bases on the DNA template: A with T, G with C, and U with A.
• RNA polymerase binds to DNA, moves along it, and links aligned ribonucleotides to create an RNA molecule.
• RNA polymerase can initiate transcription without a primer.
• Multiple RNA molecules can be transcribed from a single gene simultaneously.
• Growing RNA transcripts appear as threads extending from the DNA backbone; shorter transcripts are closer to the start of transcription, while longer ones are near the end of the gene.
• The base sequence in the RNA transcript is complementary to the template strand and the same as the non-template strand (coding strand), with U replacing T.
• The coding strand is the non-template strand of DNA.

Stages of Transcription

• Three stages: initiation, elongation, and termination.
• Initiation: Transcriptional machinery is directed to the start of the gene. The non-template DNA strand is usually shown with the 5' end on the left and the 3' end on the right. The initiation site is the first transcribed base, designated as position #1. Some promoters vary from the usual pattern, resulting in less protein production because RNA polymerase will not bind effectively.
• Two important conserved regions in promoters: -35 (TTGACAT) and -10 (TATAAT, or Pribnow box).
• Elongation: RNA polymerase continues transcribing the length of the gene.
• Termination: Transcription stops at the end of the gene.

RNA Polymerase

• RNA polymerase binds to a regulatory region upstream of the gene (5' regulatory region).
• The RNA polymerase holoenzyme includes a sigma subunit and a core enzyme.
• The core enzyme includes the NusA protein.

Directionality in Transcription

• The phosphate group on the 5' end of an entering ribonucleotide attaches to the 3' end of the growing RNA chain.
• The template strand is read by RNA polymerase from 3' to 5'.
• Polymerization occurs in the 5' to 3' direction.
• Either DNA strand can serve as the template for transcription, and genes can be transcribed in opposite directions.

Promoter

• A regulatory region located a short distance from the 5' end of a gene.
• Serves as a binding site for RNA polymerase (5' regulatory region).

Upstream/Downstream

• Upstream refers to the 5' end of the gene.
• Downstream refers to the 3' end of the gene.

RNA Polymerase Holoenzyme

• Binds to DNA, unwinds the double helix, and begins RNA synthesis.
• A multi-subunit complex composed of five subunits of the core enzyme (alpha, beta, beta', omega) plus the sigma factor.
• Scans DNA for promotor sequences.

Consensus Sequence

• Sequence that reflects the most commonly found nucleotides at each position in a group of related DNA or RNA sequences.

Sigma Factor

• Binds to the -10 and -35 regions of the promoter, melts the DNA, and dissociates after initiation.
• Alpha-helical regions of the sigma subunit contact DNA at major grooves.
• The active site contains a magnesium ion required for catalytic activity.
• Different sigma subunits can attach to RNA polymerase and will bind to different promoters.
• Causes local denaturation of the DNA duplex, forming an open promoter complex.

Transcription Bubble

• A region of single-stranded DNA within which the template strand is exposed.
• Rich in A/T base pairs.
• The sigma factor is released after initiation.
• rNTPs (ribonucleoside triphosphates) are the building blocks for RNA synthesis; "-tide" indicates the presence of a phosphate group, while "-side" indicates its absence.

Intrinsic Termination of Transcription

• The terminator sequence is about 40 base pairs long, ending in a GC-rich stretch followed by a string of six or more adenines (A's).
• The guanine (G) and cytosine (C) bases form complementary hydrogen bonds, creating a hairpin stem loop structure.
• The hairpin structure is followed by a string of about eight uracils (U's) complementary to the adenine residues on the DNA template.
• The polymerase pauses after synthesizing the uracils.
• RNA polymerase pauses if the short DNA-RNA hybrid in the transcription bubble is weak, causing it to backtrack to stabilize the hybrid.
• When the backtracking polymerase encounters the hairpin loop, it triggers the release of RNA from the polymerase and the release of the polymerase from the DNA template.

Rho Termination of Transcription

• Rho factor is a helicase protein that recognizes nucleotide sequences acting as termination signals for RNA polymerase.
• Termination sequences typically lack the string of U residues at the 3' end and usually do not have hairpin loops.
• An upstream segment called the rut (rho utilization) site is present.
• Rho binds to the nascent RNA chain at the rut site.
• Its helicase activity unwinds the DNA/RNA hybrid.
• Rho catches up to the polymerase, causing it to fall off.

Transcription in Prokaryotes vs. Eukaryotes

• Transcription is more complex in eukaryotes.
• Eukaryotes have more genes to recognize and transcribe, as well as more noncoding DNA to navigate; three different polymerases transcribe rRNA, mRNA, and small functional RNA genes.
• Gene regulation differs based on cell type, stage of development, and environment.
• Eukaryotes assemble many proteins at the promoter before transcription.
• General transcription factors (GTFs) bind before RNA polymerase II, while others bind after.
• Many accessory transcription factors and binding sites interact to regulate gene-specific expression.
• Eukaryotes have a nucleus, spatially separating transcription and translation (in prokaryotes, RNA is immediately translated).
• RNA processing is needed before mRNA leaves the nucleus.
• The primary transcript or pre-mRNA is the RNA before processing, and the fully processed transcript is called mRNA.
• The 5' end of RNA undergoes processing while the 3' end is still being transcribed.
• Eukaryotes have chromatin, unlike prokaryotes where DNA is "naked."
• Chromatin structures in eukaryotes can block RNA polymerase access to the DNA template, regulating gene expression.
• In prokaryotes, RNA is immediately translated. DNA is "naked," whereas eukaryotes have chromatin.

General Transcription Factors (GTFs)

• Bind before RNA polymerase II binds, while others bind afterward.
• Bind to sequences in the promoter or to other GTFs and attract RNA polymerase II core, positioning it at the correct site to start transcription (e.g., TFIIA and TFIIB).
• Following initiation, RNA polymerase II dissociates from most of the GTFs to elongate the primary RNA transcript.
• Some GTFs remain at the promoter to attract the next RNA polymerase.

Preinitiation Complex

• Consists of six GTFs and RNA polymerase II.

TATA Box

• A conserved region in eukaryotic promoters.
• The binding of the TATA-binding protein (TBP) is the first event in transcription.
• TBP is part of the TFIID complex, one of the six GTFs.
• TBP attracts other GTFs and the RNA polymerase II core to the promoter.

TBP (TATA-Binding Protein)

• The first event in transcription is the binding of TBP.
• Part of the TFIID complex, one of the six GTFs.
• Attracts other GTFs and the RNA polymerase II core to the promoter.

Carboxy Terminal Domain (CTD) of Polymerase II

• A protein tail subunit of RNA polymerase II.
• The CTD is phosphorylated by one of the GTFs, which weakens the connection of RNA polymerase II and permits elongation.
• The CTD is located near the site where nascent RNA emerges from the polymerase, and its amino acids are reversibly modified by phosphorylation and dephosphorylation.

Splicing and Alternative Splicing

• Splicing is the removal of introns and joining of exons, bringing exons together.
• The number and size of introns vary from gene to gene and from species to species.
• Alternative splicing involves producing different mRNAs and proteins from the same primary transcript by splicing together different combinations of exons.
• Mutations with serious consequences for the organism can result from splicing defects.
• Proteins made by alternative splicing are usually related.

snRNPs (Small Nuclear Ribonucleoproteins)

• snRNAs are complementary to the consensus sequences at splice junctions.
• Five snRNPs are complexes of protein and one or five snRNAs.
• The spliceosome consists of snRNPs and additional proteins that remove introns and join exons.
• snRNPs interact with the CTD.

RISC/Dicer

• RISC (RNA-Induced Silencing Complex): Unwinds double-stranded RNA into the biologically active single-stranded miRNA. RISC consists of RNA and Argonaute. Argonaute is an endonuclease that degrades RNA.
• Dicer: An enzyme that recognizes double-stranded RNA (dsRNA) molecules and cleaves them into ~22-nucleotide products.

RNA Interference (RNAi)

• A process that silences specific genes by degrading their mRNA.
• Used to reduce the expression of mutated or harmful genes.

Protein Structure - Levels of Organization

• Proteins have multiple levels of structural organization that dictate their function.

Amino Acids

• There are 20 amino acids, requiring at least a three-letter code in mRNA to specify them (4 × 4 × 4 = 64 possible codons).
• Excess codons lead to degeneracy of the genetic code.
• The genetic code is degenerate, meaning that some amino acids are specified by two or more different triplets.

Alpha Helix

• A common secondary structure in proteins.

Beta Sheet

• Another common secondary structure in proteins.

Enzymes

• Kinases: Enzymes that attach phosphate groups to the hydroxyl groups of the amino acids serine, threonine, and tyrosine.

Genetic Code

• One-gene–one-polypeptide hypothesis: Proposed by Beadle and Tatum in 1945, stating that each gene encodes a single polypeptide, which may function independently or as a subunit of a more complex protein.

Stop Codons

• Codons that do not specify an amino acid, signaling the end of translation.

Directionality in Translation

• tRNA binds 3' to 5' and reads mRNA from 5' to 3'.

tRNA Structure

• Has an L-shaped folded cloverleaf structure, determined by X-ray crystallography.
• All tRNAs fold into virtually the same L-shaped conformation except for differences in the anticodon loop and aminoacyl end.

Aminoacyl-tRNA Synthetase

• Enzymes that attach tRNAs to their corresponding amino acids.

Wobble

• The ability of a tRNA to bind to several alternative codons through a loose kind of base pairing at the 3' end of the codon and the 5' end of the anticodon.
• The third nucleotide of an anticodon (at the 5' end) can form multiple alignments.
• "Wobble rules" dictate which nucleotides can and cannot form hydrogen bonds with alternative nucleotides through wobble.

Ribosome Structure

• Ribosomes are composed of a small subunit and a large subunit.

Small Subunit/Large Subunit

• In prokaryotes, the small subunit is 30S, and the large subunit is 50S, forming a 70S ribosome.
• In eukaryotes, the small subunit is 40S, and the large subunit is 60S, forming an 80S ribosome.

Shine-Dalgarno Sequence

• (Prokaryotes only) special sequences that pair with the 3' end of rRNA called the 16S rRNA, in the 30S ribosomal subunit.
• Positions the initiator codon in the P site where the initiator tRNA will bind.
• mRNA can pair only with a 30S subunit that is dissociated from the rest of the ribosome.

Initiation Factors

• Required for correct initiation of translation (e.g., IF1, IF2, IF3).
• IF3 keeps the 30S subunit dissociated from the 50S subunit.
• IF1 and IF2 ensure that only the initiator tRNA enters the P site.
• The 30S pre-initiation complex consists of the 30S subunit, mRNA, and initiator tRNA.

Initiation Complex

• Formed at the 5' end of the mRNA and consists of initiation factors, the cap structure, the 40S subunit, and the initiator tRNA.

Translation Steps

• The steps are: initiation, elongation, and termination.
• Elongation takes place inside the transcription bubble.
• Prokaryotic mRNA can be translated at the 5' end while the 3' end is still being synthesized.
• Eukaryotic mRNA needs to undergo more processing to be translated, involving adding caps at the 5' end, splicing to eliminate introns, and adding a 3' tail of adenine nucleotides (polyadenylation).

EF-Tu & EF-G

• Elongation factor Tu (EF-Tu) and elongation factor G (EF-G) assist the elongation process.
• EF-G fits into the A site.

Release Factors

• Termination of elongation depends on eukaryotic release factors (eRF).
• In eukaryotes, eukaryotic release factor eRF-1 recognizes all three termination codons (UAA, UAG, and UGA) and, with the help of protein eRF, terminates translation.
• Upon termination, the ribosome is disassembled, and the completed polypeptide is released.
• RF1, 2, and 3 recognize stop codons in prokaryotes.

Phosphorylation

• A post-translational modification in which an amino acid residue (serine, threonine, or tyrosine) is phosphorylated by a protein kinase with the addition of a covalently bound phosphate group (requires ATP).
• Kinases attach phosphate groups, while phosphatases remove them.
• Phosphate groups are negatively charged, changing protein conformation.
• Reversible switch to control enzyme activity, protein/protein interactions, and protein-DNA interactions.

Ubiquitination

• Targets the protein for degradation.
• Ubiquitin is a protein found in all eukaryotes.
• The 26S proteasome is a protease recruited by the addition of ubiquitin to the amino acid lysine (ubiquitination).
• Short-lived proteins, such as cell cycle regulators, and damaged proteins are targeted for destruction by ubiquitination.

Protein Targeting

• Some proteins are destined for the nucleus, including RNA/DNA polymerases and transcription factors.
• These proteins have a nuclear localization sequence (NLS).

Nuclear Localization Sequence (NLSs)

• Amino acid sequences embedded in the surface of nucleus-bound proteins.
• Recognized by importin (cytoplasmic receptor proteins) that transport newly synthesized proteins through nuclear pores.

30S + 50S vs. 40S + 60S

• 30S + 50S ribosomes are prokaryotic and have a Shine-Dalgarno sequence.
• 40S + 60S ribosomes are eukaryotic and use a scanning model.
• A site (aminoacyl) binds incoming aminoacyl-tRNA whose anticodon matches the codon in the A site of the 30S subunit.
• P site (peptidyl) is where the next codon interacts with the anticodon of tRNA in the 30S subunit. It binds the growing peptide chain, part of which fits into the tunnel structure of the 50S subunit.
• E site (exit) contains deacetylated tRNA that is ready to be released from the ribosome.
• An initiation complex and associated factors (IFs), elongation factors (EFs), and release factors (RFs) are required for translation.

Other Definitions

• Monocistronic: Eukaryotic mRNA molecules encode a single polypeptide.
• Polycistronic: Bacterial mRNA encodes several different polypeptide molecules.
• Promoter: A regulatory region a short distance from the 5' end of a gene, acting as a binding site for RNA polymerase.
• 5' Untranslated Region (5' UTR): An intervening part at the 5' end of mRNA.
• 3' Untranslated Region (3' UTR): Transcription of a gene continues beyond the protein-encoding segment, creating a 3' end.
• Termination: RNA polymerase recognizes special nucleotide sequences that act as a signal for chain termination (initiates the release of RNA and enzyme).
• Two major release mechanisms for termination in E. coli: intrinsic and rho-dependent.
• Polymerase A: Makes the poly-A tail on the 3' end of mRNA.
• Polyadenylation Signal: AAUAAA is recognized by polymerase A, which cuts off the end of the RNA ~20 bases further down.
• GU-AG rule: Introns have GU at the 5' end and AG at the 3' end.
• Gene Silencing: Reducing or eliminating the production of protein from its corresponding gene.
• Huntington Disease (HD): A mutation in the HTT gene causes the toxic accumulation of huntingtin protein in neurons, leading to motor dysfunction, cognitive decline, and psychiatric issues. RNAi can help by targeting and degrading HTT mRNA, reducing huntingtin protein production, and slowing/preventing neuronal damage.
• N-Terminus and C-Terminus: The ends of a polypeptide chain.
• D-Loop (Dihydrouridine Loop): Binds aminoacyl-tRNA synthetase to accept new amino acids.
• TΨC Loop: Facilitates the binding of tRNA to the A site in the ribosome.
• Acceptor Stem (3' end): The attachment site for the appropriate amino acid, recognized and bound by aminoacyl-tRNA synthetase, ensuring the right amino acid is attached to the tRNA.
• Decoding center: The function of the 30S subunit is to ensure that only tRNAs carrying anticodons that match the codon (cognate tRNAs) will be accepted into the A site.
• Peptidyltransferase center: The function of the 50S subunit is where peptide bond formation is catalyzed.
• Polyribosomes: During translation, multiple 70S units (ribosomes) act simultaneously.
• Coupled transcription-translation: Translation happens at the same time as transcription.
• Free ribosomes: Make proteins used inside the cell.
• Bound ribosomes: Make proteins secreted outside the cell or used in lysosomes (involved in digestion bacteria and "worn out" cell parts).
• Genome: The entire set of genes in the genome.
• Transcriptome: The complete set of coding and noncoding transcripts in an organism, organ, tissue, or cell.
• Proteome: The complete set of proteins in an organism, organ, tissue, or cell.
• Isoforms: Multiple proteins with different combinations of functional domains.

Extra Notes

• rRNA plays an essential role in the translation of proteins.
• siRNA is involved in repressing RNA from exogenous sources (e.g., viral RNA).
• snRNA participates in the eukaryotic molecular machine that removes introns from RNA transcripts (e.g., the spliceosome).

Practice Questions

• Which region will be transcribed into RNA? Region A
• Based on the sequences of the DNA strands shown, the ___ strand will be used as the template strand, and RNA polymerase will move from ______: the top strand will be the template strands, and RNA polymerase will move from right to left.
• The predicted transcript will start with the sequence _____: 5'GCCAUUAGU3'
• If the mRNA encoded by this sequence was translated from left to right starting at the first ribonucleotide, the amino acid sequence of the polypeptide would be: Met-Phe-Glu-Trp
• In eukaryotes, pre-mRNA-processing enzymes or factors: All of the above.
• Which of the following does NOT describe a difference between prokaryotes and eukaryotes?: Transcription initiation in eukaryotes requires a promoter, while prokaryotes transcribe all genes. This statement is incorrect.
• The most important DNA sequence or "element" in the promoter region of eukaryotic genes, which helps to initiate transcription (and to which TBP binds), is known as the: TATA box
• Which statement best describes the main distinction between the two classes of small regulatory RNAs: siRNA and miRNA?: siRNAs target transcripts from exogenous sources; miRNAs target transcripts from the cell's own genes.
• Which region of a tRNA molecule is responsible for binding to a specific amino acid?: The acceptor stem/3' end.
• Which structural feature of tRNA is responsible for binding to the appropriate aminoacyl-tRNA synthetase enzyme?: Anticodon loop.
• The A position of ribosomes is the site where: mRNA is decoded by tRNA and the corresponding amino acid is recruited.
• Which of the following is not part of the 30S pre-initiation complex in prokaryotes?: 50S subunit. The 30s Pre-initiation complex is made of the mRNA, initiator tRNA and initiation factors (IF1, IF2 and IF3).
• During the elongation phase of translation, the aminoacyl-tRNA carrying the next amino acid to be added to a growing polypeptide chain: First binds to the A site in the ribosome.
• When translation machinery encounters a "stop" codon: Translation is terminated by release factors.
• You have discovered a new single-celled organism, is it a prokaryote or eukaryote? The sedimentation coefficient of ribosomes is 70S (50S and 30S subunits): Prokaryote.
• The human genome contains ~20,000 protein-coding genes, but the proteomes of many cells can have more than 100,000 different proteins. A possible explanation is that: alternative splicing and post-translational modification of proteins increases the number of proteins the cells can produce.