RNA Structure and Classes

RNA Structure and Classes

• DNA has one hydroxyl group at the 3' position, while RNA has two at the 3' and 2' positions
• RNA contains uracil, whereas DNA contains thymine
• RNA is typically single-stranded; DNA is typically double-stranded
• Secondary structures in RNA, such as hairpins, are formed by inverted repeats, contributing to RNA shape which is helpful for RHO-independent termination
• rRNA is present in both prokaryotes and eukaryotes, found in the cytoplasm, and contributes to structure and function
• mRNA exists in both prokaryotes and eukaryotes, located in the cytoplasm and nucleus, and carries the genetic code for proteins
• tRNA is present in both prokaryotes and eukaryotes, found in the cytoplasm, and helps incorporate amino acids into polypeptide chains
• snRNA is exclusively found in eukaryotes, specifically in the nucleus, and is responsible for processing pre-mRNA
• snoRNA is exclusively found in eukaryotes, specifically in the nucleus, and processes and assembles mRNA

Introduction to Transcription

• Transcription requires: a DNA template, raw materials (rNTPs), and a transcription apparatus (RNA polymerase and accessory proteins)
• Ribonucleotide triphosphates (rNTPs) serve as the building blocks for transcription
• The transcription apparatus includes RNA polymerase, which is essential, and accessory proteins
• Transcription does not need a primer
• Newly synthesized RNA is antiparallel and complementary to the template DNA
• The template strand is read in the 3' to 5' direction
• The newly synthesized RNA is made in the 5' to 3' direction
• The sense strand is the non-template strand, while the antisense strand is template strand.
• The template strand is used to make RNA; the non-template strand is not used
• The coding strand is the sense strand, and the non-coding strand is the nonsense strand, which is the template strand
• Different genes can be transcribed from either DNA strand, but individual genes are transcribed from only one strand
• The promoter is a nucleotide sequence that signals the start of transcription
• A coding region is transcribed into RNA by RNA polymerase
• The terminator is a nucleotide sequence that signals the end of transcription
• The transcription start site is designated as +1
• Consensus sequences are located at -10 and -35 upstream
• Upstream is to the left, downstream is to the right relative to the direction of RNA synthesis
• Consensus sequences represent the most frequent nucleotides at each position in DNA

Bacterial Transcription

• Bacterial promoters have consensus sequences at -35, -10, and an upstream element
• Spacing of consensus sequences is important for initiating transcription
• The core RNA polymerase is a multi-subunit enzyme with two alpha, one beta, one beta prime, and one omega enzyme and is key in RNA synthesis
• The holoenzyme comprises the core RNA polymerase and a sigma factor
• The sigma factor helps RNA polymerase bind to DNA for transcription initiation
• Sigma factor associates to form a holoenzyme
• Holoenzyme binds to the -35 and -10 consensus sequences in the promoter.
• Holoenzyme binds to the promoter tightly and unwinds the double stranded DNA
• A rNTP complimentary to the base at the start site is the first nucleotide in the RNA
• Two phosphate groups are cleaved from each rNTP creating an RNA nucleotide that is added to the 3' end of the growing RNA
• The sigma factor is released as the RNA polymerase moves beyond the promoter
• Abortive initiation makes and releases short transcripts (2-6 nt long) while the enzyme is still bound to a promoter
• The bacterial transcription bubble is ~18 nt in size
• The rate of transcription is ~40 nt/sec
• Phosphodiester bonds form between the ribonucleotides in the RNA strand
• RNA polymerase pauses or backtracks during elongation due to sequence-dependent signals, proofreading, and gene regulation

Rho-Dependent Termination

• The Rut site is where Rho binds and moves toward the 3' end
• Rho protein uses helicase activity to unwind the DNA-RNA hybrid, stopping transcription

Rho-Independent Termination

• Inverted repeats induce RNA to form a hairpin, destabilizing DNA-RNA pairing
• A poly-adenine stretch has only adenine nucleotides at the end
• During active transcription, the inverted repeats causes RNA folding and the poly-adenine stretch destabilizes the DNA:RNA pairing separating from DNA

Eukaryotic Transcription

• Chromatin structure must be modified for eukaryotic transcription for DNA accessibility
• Chromatin-remodeling complexes use ATP hydrolysis to alter chromatin
• Histone tail modifications affect chromatin structure
• RNA polymerase I makes larger RNAs
• RNA polymerase II makes pre-mRNA, some snRNAs, snoRNAs, and miRNAs and is key in producing mRNA that is translated into protein
• RNA polymerase III makes tRNAs, small RNAs, some snRNAs, and some miRNAs
• The TATA box sequence is TATAAA, located at -25, and serves as a recognition site for TATA-binding protein to initiate transcription by RNA polymerase II
• With respect to the transcriptional start site, the core promoter is immediately upstream, the regulatory promoter is upstream past the TFIIB recognition element, and enhancers are located upstream

Transcription Factors

• General transcription factors bind to the TATA box first
• TATA-binding protein (TBP) is a general transcription factor that binds to the TATA box to initiate transcription
• The basal transcription apparatus includes RNA polymerase II, general transcription factors (TFIIA, TFIIB, TFIID, TFIIF, TFIIH), and promoter DNA
• The mediator acts as a bridge between transcription factors and RNA polymerase II to control gene expression
• During the elongation stage of eukaryotic transcription, DNA is unwound, RNA is synthesized, and RNA translocates along the template while maintaining separation from DNA and a transcription bubble
• RatI is an exonuclease that attaches to the 5' end of the trailing RNA to terminate transcription when it reaches the polymerase

Eukaryotic pre-mRNA Processing

• mRNA has a 5' UTR, a protein-coding region (with start and stop codons), and a 3' UTR
• AGGAGG (Shine-Dalgarno) are ribosome binding sites found in the 5′UTR of prokaryotes
• The 3' UTR affects mRNA stability and regulates translation of mRNA protein-coding sequences
• Pre-mRNA still has proteins attached and yields mRNA after processing
• Pre-mRNA processing occurs in eukaryotes but not in bacteria because of no nucleus present separating translation and transcription in bacteria
• The addition of the 5' cap (7-methylguanosine with a 5' to 5' bond and methyl groups on the 2' OH of the next two nucleotides) facilitates ribosome binding, increases mRNA stability, and enhances RNA splicing
• The 5' cap signals the start site of transcription
• The 3' poly(A) tail increases mRNA stability, aids in export from the nucleus, and facilitates ribosome binding
• The 3' poly(A) tail addition uses the AAAAAUAAAAU sequence and adds adenine nucleotides to the 3' end of pre-mRNA
• The 3' poly-A tail determines where cleavage occurs
• Bacterial genes are colinear to proteins, but eukaryotic genes aren't as they only have 5′ and 3′ ends that doesnt encode whereas eukaryotes have many regions that dont encode
• Introns are sequences in mRNA not encoded into protein that are spliced and degraded
• Exons are sequences in mRNA that encodes for protein
• Spliceosomal introns consist of group II introns

Splicing of Eukaryotic pre-mRNA

• Splicing occurs inside the nucleus
• Splicing requires a 5' splice site, a branch point (adenine nucleotide 18-40 nt upstream of the 3' splice site), and a 3' splice site
• The spliceosome is made up of macromolecules

Splicing Terms

• snRNAs are inside the spliceosome, associates with protein to form snRNPs
• snRNPs consists of snRNA five snRNPs, U1, U2, U4, U5, U6
• A lariat is a loop that introns form, formed when being spliced and degraded
• Alternative processing allows a single pre-mRNA to be processed differently to produce alternative RNA types and proteins
• Alternative splicing is controlled by proteins and ribonucleoproteins that bind to sites within introns and exons and determines which splice and cleavage sites are used

RNA Cleavage Sites

• Multiple 3' cleavage sites allow cleavage and polyadenylation at different sites, producing mRNAs of different lengths
• Calcitonin example, human gene (6 exons, 5 introns) is transcribed into pre-mRNA has two possible 3' cleavage:
• In the thyroid gland cells have a 3'cleavage and polyadenylation after exon 4 being translated into calcitonin regulates calcium concentration in blood
• In brain cells pre-mRNA identical only 3' occurs after exon 6, splicing exon 4 and removed it, the protein makes causes dilation ofblood vessles and can fxn in transmission of pain. some suggest related to migraines

tRNA, rRNA, and Ribosome Processing

• tRNA is a link between the genetic code in mRNA and the amino acids
• The anticodon on tRNA pairs with the codon on mRNA allowing amino acids to be added to a growing polypeptide chain at positions specified by genetic instructions

tRNA Structure

• Tertiary structure has L shape-on the 3' end is the acceptor arm where AA attaches always (CCA)-the bottom of structure is the anticodon site
• Cloverleaf structure with 4 arms; L-shaped tertiary structure
• Anticodon and amino acid attachment site (3' end always is CCA) location

Ribosome Structure

• Consists of rRNA plus protein
• Ribosomes in bacteria : large (50S) - subunits 70S - ribosome size small (60S) - subunits, in eukaryotes : large (60S) - subunits (80S) - ribosome size (40S) - small

rRNA

• Precursor prokaryotic rRNA transcription is methylated, methyl groups inside, intermediates and tRNA separates segments to mature mRNA has 5S rRNA
• Precursor eukaryotic rRNA transcription is methylated,segments without tRNA that is encoded different in each cell Small nucleolar RNAs (snoRNAs) helps cleave and process the rRNAs for assembly into ribosomes in eukaryotic cells
• snoRNP associates with proteins to form