BIOMG 1350 Lecture 3 - RNA

RNA

Monomers in RNA are still nucleotides or ribonucleotides.

RNA's ribose sugars contain an OH group on the 2' carbon, distinct from DNA's hydrogen on the 2' carbon. This means that RNA is a much more unstable molecule than DNA.

A single-stranded nucleic acid that plays key roles in converting genetic information from DNA into proteins. RNA is made of nucleotides with a ribose sugar, a phosphate group, and nitrogenous bases (adenine, uracil, cytosine, guanine). Types include mRNA (carries genetic code), tRNA (transfers amino acids), and rRNA (forms ribosomes).

Nucleotides in RNA form polymers by phosphodiester linkage via a condensation (dehydration synthesis) reaction, and these nucleotides are joined at the same places as DNA (the 3' OH group on the ribose sugar and the 5' carbon on the ribose sugar too).

RNA is usually single stranded and it contains a negatively charged backbone of alternating ribose sugars and phosphate groups connected via phosphodiester linkages.

Has a 5' end (with a phosphate) and a 3' end (with a free OH) just like DNA.

RNA contains what base not present in DNA? What are its characteristics?

RNA contains Uracil in the place of Thymine in DNA. Thymine and Uracil can both form 2 hydrogen bonds with Adenine.

Uracil is a pyrimidine.

Uracil is used instead of Thymine as thymine is less reactive and more stable for housing genetic information whereas Uracil is needed for the other uses of RNA which do not include maintaining genetic info like DNA.

Multifaceted Functions of RNA

Single stranded RNA molecules can fold into 3D structures in a similar fashion as proteins. This is achieved through base pairing within the same RNA molecule.

RNA can fold into ribozymes or RNA enzymes that catalyze reactions in the cell.

RNA’s different functions can be seen in the 3 main types: tRNA, rRNA, and mRNA

What are the advantages for cells in making RNA? Why is RNA single stranded despite having so many DNA like properties?

Making enzymes, different protein variants via alternative splicing, can control amount of protein, genetic information is safely copied rather than altered meaning genetic info in DNA can stay in the nucleus rather than go into the cytoplasm, etc.

The reason RNA is single stranded but is chemically very similar to DNA is just because RNA polymerase synthesizes a single stranded RNA molecule that compliments the DNA template during transcription.

Single stranded RNA is also able to form complex structures with various functions, something that would be impossible if RNA was not single stranded.

Promoter

A DNA sequence located near the start of a gene where RNA polymerase and transcription factors bind to begin transcription.

Key points:

• Determines which genes are expressed and how often.

• Found upstream (5′ direction) of the coding region.

• Acts as a control switch for gene expression.

The position and orientation of the promoter determines which strand of the DNA is the template vs coding strand and where RNA synthesis starts.

Poly-A Tail

A string of adenine (A) nucleotides added to the 3′ end of eukaryotic mRNA after transcription.

Key points:

• Protects mRNA from degradation.

• Helps export mRNA from the nucleus to the cytoplasm.

• Assists in translation by helping ribosomes recognize the mRNA.

5′ Methyl Cap (5′ Cap)

A modified guanine nucleotide added to the 5′ end of eukaryotic mRNA shortly after transcription.

Key points:

• Protects mRNA from degradation by exonucleases.

• Helps ribosomes recognize the mRNA for translation.

• Assists in nuclear export of the mRNA.

Transcription

The process in which a DNA sequence is copied into a complementary RNA sequence. In protein-coding genes, this produces messenger RNA (mRNA), which carries the genetic instructions from DNA to the ribosome. Occurs in the nucleus in eukaryotes and the cytoplasm in prokaryotes. Transcription is catalyzed by RNA polymerase.

• Initiation

  • RNA polymerase binds to a promoter region on the DNA.

  • The DNA strands unwind near the start site of the gene.

  • The template strand is exposed for RNA synthesis.

• Elongation

  • RNA polymerase moves along the DNA template strand in the 3’ → 5’ direction.

  • Complementary RNA nucleotides are added to the growing RNA strand in the 5’ → 3’ direction.

  • Base-pairing rules apply: A → U, T → A, C → G, G → C.

• Termination

  • RNA polymerase reaches a termination sequence in the DNA.

  • The RNA transcript detaches from the DNA template.

  • In eukaryotes, the transcript is initially pre-mRNA and undergoes processing (5’ cap, poly-A tail, splicing) to become mature mRNA.

Template Strand

The DNA strand that is used as a pattern during transcription to synthesize a complementary RNA molecule.

• RNA nucleotides pair with the template strand using base-pairing rules (A → U, T → A, C → G, G → C).

• The RNA is antiparallel to the template strand (RNA grows 5’ → 3’, template read 3’ → 5’).

• Only one strand of DNA serves as the template for a given gene, though the opposite strand can serve as the template for other genes.

Note: The complementary DNA strand that is not transcribed is called the coding strand because its sequence matches the RNA (except T → U).

Terminator

A DNA sequence that signals the end of transcription.

• In bacteria, the terminator sequence is transcribed into RNA, which causes RNA polymerase to detach and release the RNA transcript.

• In eukaryotes, RNA polymerase II transcribes a polyadenylation signal (AAUAAA), after which proteins cut the pre-mRNA downstream to release it.

• Marks the downstream boundary of a transcription unit.

General Transcription Factors and the Transcription Initiation Complex

General transcription factors are proteins in eukaryotes that help RNA polymerase II bind to the promoter and start transcription.

• Some recognize specific sequences, like the TATA box.

• They position RNA polymerase in the correct orientation on the DNA.

• Essential for gene regulation.

• They are only in eukaryotes.

The assembled complex of RNA polymerase and general transcription factors bound to the promoter.

• Unwinds the DNA and begins RNA synthesis at the transcription start site.

• Ensures accurate initiation of transcription.

Pre-mRNA (primary transcript)

The initial RNA transcript synthesized from a protein-coding gene in eukaryotes during transcription. It contains both exons (coding regions) and introns (noncoding regions). Before leaving the nucleus, pre-mRNA undergoes processing to become mature mRNA:

1. 5’ cap added – protects the RNA and helps ribosome binding.

2. Poly-A tail added at 3’ end – increases stability and aids export from the nucleus.

3. Splicing – introns are removed, and exons are joined together.

Pre-mRNA is essentially the raw copy of a gene’s instructions before it’s finalized for translation.

The genome is the name for… ?

Collection of all genes

A gene is a segment of DNA that contains the instructions for the manufacture of RNA molecules

How do cells know where to start and which DNA sequences to transcribe into DNA?

Start of transcription is defined by a promoter. A promoter is a region of DNA that contains specific sequences that are recognized by RNA polymerase with the help of transcription factors (in this case sigma factor) to start transcription.

Sigma factor in prokraryotes allows the RNA polymerase to recognize the promoter and general transcription factors do the same thing in eukaryotes.

RNA polymerase is designed in a way such that it can only orient itself on the DNA in a specific way such that it moves from the 3' to 5' direction synthesizing from the 5' to 3' direction.

Sigma Factor

Sigma factor is embedded within RNA polymerase and it’s the specific part of the RNA polymerase that allows the RNA polymerase to recognize the promoter.

Sigma factors are exclusively present in prokaryotes.

Coding Strand

The coding strand is the DNA strand whose sequence matches the mRNA (with T instead of U). It's not directly copied by RNA polymerase — that's the job of the complementary template strand, which polymerase reads to build the mRNA.

Resulting RNA sequence formed through transcription is exactly the same as the coding strand though every T is replaced with U.

Spliceosome

A large molecular complex in eukaryotic cells made of small nuclear RNAs (snRNAs) and proteins that removes introns from pre-mRNA and joins exons together to form mature mRNA. It ensures that only the coding sequences are included for translation.

Basically, it’s the cell’s RNA editor and engages in RNA splicing or RNA curating.

Exons and Introns

DNA sequences within a gene that code for proteins or functional RNA and are retained in the final mRNA after RNA processing.

DNA sequences within a gene that do not code for proteins and are removed from the pre-mRNA during RNA splicing.

Ribozymes

RNA molecules that act as enzymes, catalyzing chemical reactions. They can self-splice introns, cleave or join RNA, and even help form peptide bonds in the ribosome. They act as enzymes because their three-dimensional RNA structure allows them to fold into specific shapes that position reactants precisely, similar to how protein enzymes work.

This shows that RNA can be both genetic material and a catalyst, supporting the “RNA world” idea in evolution.

Alternative RNA Splicing

A process in eukaryotic cells where a single pre-mRNA can be spliced in different ways to produce multiple mRNA variants. This allows one gene to encode different proteins by including or excluding certain exons. Many genes are known to give rise to two or more different polypeptides, depending on which segments of that gene are treated as exons during RNA processing.

It’s a key mechanism for increasing protein diversity without increasing the number of genes.

RNA Polymerase

In contrast to DNA polymerase, RNA polymerase can initiate the synthesis of complementary nucleotides on the template strand without a primase.

The enzyme that synthesizes pre mRNA by adding nucleotides complementary to a DNA template strand.

• Works 5’ → 3’, adding nucleotides to the 3’ end of the growing RNA.

• Unlike DNA polymerase, it does not need a primer to start synthesis.

• In eukaryotes, RNA polymerase II transcribes pre-mRNA, polymerase I and III synthesize standalone RNA molecules.

• In bacteria, a single RNA polymerase transcribes all types of RNA.

Primase is a form of RNA polymerase used in DNA replication.

Accuracy of RNA Polymerase verses DNA Polymerase

Because RNA polymerase does not need a primase, this leads to RNA polymerase being much less precise and accurate compared to DNA polymerase. However this isn't as big of a problem as RNA can be degraded and doesn’t permanently affect the genome.

How do cells know where to stop transcribing the DNA sequence into RNA?

RNA polymerase stops after the terminator, then it falls off from the DNA and then the completed RNA is released.

Terminator sequence is included in the transcribed RNA, though anything after the Terminator is not transcribed into the RNA.

What does a cell do if it needs multiple copies of the same mRNA?

Many RNA polymerases can transcribe a single gene simultaneously. Before one RNA polymerase finishes transcribing a gene, another RNA polymerase can start.

Types of RNA

Some examples of non-protein coding RNA are: rRNA, tRNA, miRNA, piRNA, siRNA, etc.

Three main different types of RNA: mRNA, tRNA, rRNA which all work together in the process of protein synthesis.

RNA Sizes

mRNA (100 to 10 kb), rRNA (100-5000 b), tRNA (70-90 b), and there are many others.

mRNA (mature)

Transcribing genes into mRNA allows cells to amplify the expression of certain genes thereby amplifying the number of proteins produced. (1 gene -> 5 mRNA -> 25 proteins).

Transcribing genes into mRNA also allows cells to regulate the expression of certain genes.

Transcription can be regulated to change cell behavior by transcription factors or regulators.

tRNAs

Contain an anticodon in one of their loops. This anticodon allows the mRNA to be bound by the tRNA, and the tRNA carries a specific amino acid.

Adapter that translates the codon to a specific amino acid that is incorporated during protein synthesis.

tRNA is connected to its proper amino acid via enzymes called amino acyl-tRNA synthetases.

For every codon that specifies an amino acid, there is a tRNA adaptor that can recognize that codon through base-pairing as it has a complementary ‘anticodon’ sequence in it.

How do cells couple the right amino acid to tRNA?

Enzymes called amino acyl-tRNA synthetases attach the correct amino acid to the appropriate tRNA. For example, the codon UGG specifies the amino acid tryptophan. Tryptophan becomes linked to a tRNA with a CCA anticodon.

There is at least one amino acyl-tRNA synthetase for each amino acid and each amino acyl-tRNA synthetase is specific for one amino acid and appropriate tRNA

Ribosomes and rRNA

mRNA is decoded on ribosomes and this process is called translation

A ribosome contains EPA (exit site, peptidyl tRNA site, and aminoacyl - tRNA site) and is composed of a large ribosomal and small ribosomal subunit

• Large ribosomal subunit (catalyzes peptide bond formation)

• Small ribosomal subunit (binds mRNA and matches tRNAs to mRNA codons)

The complete ribosome contains 4 rRNA molecules + 82 proteins

rRNA folds to create ribosomes, and thus ribosomes are a combination of RNA and proteins

Capping and polyadenylation

mRNA undergoes capping and polyadenylation

• A 5' cap is put on the 5' end of the mRNA almost immediately as the mRNA is being transcribed

• A poly A tail is put on the 3' end of the mRNA

• These modifications can affect how quickly the mRNA is degraded (mRNA stability) and how efficiently the mRNA is translated

• 5' Capping -> splicing -> 3' Polyadenylation

Translation

The process by which the sequence of codons in mRNA is decoded to assemble a polypeptide (protein). Occurs at ribosomes in the cytoplasm. tRNA molecules bring specific amino acids to the ribosome, matching their anticodon to the mRNA codon, linking amino acids in the correct order. Translation has three main stages: initiation, elongation, and termination.

• Initiation

  • The small ribosomal subunit binds to the mRNA near the start codon (AUG).

  • The initiator tRNA carrying methionine (Met) pairs its anticodon with the start codon.

  • The large ribosomal subunit joins to form a complete ribosome, with tRNA positioned at the P site.

• Elongation

  • A tRNA carrying the next amino acid binds to the A site of the ribosome, matching its anticodon to the mRNA codon.

  • A peptide bond forms between the amino acid on the tRNA in the P site and the amino acid on the tRNA in the A site.

  • The ribosome shifts (translocates) one codon down the mRNA:

    • The tRNA in the P site moves to the E site and exits.

    • The tRNA in the A site moves to the P site, leaving the A site open for the next tRNA.

  • This cycle repeats, lengthening the polypeptide chain.

• Termination

  • The ribosome reaches a stop codon (UAA, UAG, or UGA).

  • Release factors bind to the stop codon.

  • The polypeptide chain is released, and the ribosomal subunits dissociate from the mRNA.

  • Start codon always codes for an amino acid but the stop codon does not code for an amino acid.

How many possible variations with a 3 base codon? Can multiple codons code for the same amino acid

A 3-base code (a ‘codon’) specifies amino acids. Since there are 64 possible variations in a 3-

base code, most amino acids are encoded by multiple codons.

Human Genome Contains… ?

6.4 billion base pairs per diploid genome

20,000 protein coding genes, which only make up about 1% of the DNA

5,000 (non protein) RNA coding genes

Most mRNA is dedicated to protein coding whereas protein coding genes do not make up majority of DNA