Lab 3. Review of the Central Dogma

Coding strand

The strand of DNA that has the same sequence as the mRNA (except T instead of U); during transcription, this strand is NOT used as the template

Template strand (non-coding strand)

The strand of DNA that RNA polymerase uses as a template to synthesize RNA; complementary to both the coding strand and the mRNA

RNA polymerase

The enzyme that separates DNA strands and adds ribonucleoside triphosphates complementary to the non-coding template strand; synthesizes RNA in the 5' to 3' direction

Promoter

A special region of DNA immediately preceding a gene that directs RNA polymerase where to begin transcription; contains common elements like the TATA box

TATA box

A TA-rich DNA sequence located about 25-30 nucleotides upstream of the transcription start site; found in promoters of genes transcribed by RNA polymerase II

General transcription factors

Proteins that guide RNA polymerase to the promoter region; contain a large complex that binds RNA polymerase and helps initiate transcription

Upstream

Locations 5' to a given site on DNA; sequences that come before the transcription start site

Downstream

Locations 3' to a given site on DNA; sequences that come after the transcription start site

Terminator

A DNA sequence that signals where RNA synthesis should stop; causes RNA polymerase to terminate transcription

Pre-RNA

The initial RNA transcript before processing; contains a 5' cap, a polyA tail at the 3' end, and all transcribed RNA including introns; remains in the nucleus

5' cap

A modified guanine added to the 5' end of the pre-RNA transcript; added during RNA processing

PolyA tail

A string of adenine nucleotides added to the 3' end of the pre-RNA transcript; added during RNA processing

Introns

Sequences that are transcribed but later spliced out of the pre-RNA; not included in the mature mRNA; may contain elements that control expression

Exons

Sequences that are transcribed and preserved after splicing; encode amino acid sequence of the protein

Mature mRNA

mRNA after introns have been spliced out; contains exons, 5'UTR, and 3'UTR; exported from the nucleus for translation

Untranslated Regions (UTRs)

Regions at the 5' and 3' ends of the mature mRNA that are transcribed but not translated; important for mRNA localization and stability

Splicing

The process of removing introns from pre-RNA and joining exons together; occurs in the nucleus before mRNA is exported

Translation

The process where the mRNA nucleotide sequence is converted into an amino acid polypeptide sequence; occurs systematically with every three nucleotides (codon) specifying one amino acid

Codon

A sequence of three nucleotides in mRNA that specifies a particular amino acid or signals termination of translation

Start codon

The nucleotide sequence AUG (encoding methionine) that signals the start of translation

Stop codons

Three codons (UAA, UAG, UGA) that do not add an amino acid but cause termination of translation

Wobble base

The third nucleotide in a codon; can most often be changed without changing the identity of the amino acid due to redundancy in the genetic code

Reading frame

One of three possible ways to read a single mRNA sequence; each frame produces a different polypeptide sequence. For DNA, there are 6 possible reading frames (3 forward, 3 reverse)

Frameshift mutation

A mutation that results in insertion or deletion of nucleotides, changing the reading frame; produces a completely different protein and often a premature stop codon

Cis-control elements (enhancers)

DNA sequences (often called enhancers) where transcription factors bind to stimulate or inhibit transcription; located on the same chromosome as the gene they regulate

Enhancer location

Can be found upstream of the transcription start site (sometimes up to 1 megabase), within introns, or downstream of genes; number and location vary by gene

Poly(A) site

A site at the end of the last exon that regulates cleavage of the RNA, thereby controlling translation; where the polyA tail is added

Mutation types affecting translation

Frameshift mutations (insertions/deletions) change reading frame; nonsense mutations create premature stop codons; missense mutations change single amino acids

Genetic code redundancy

Most amino acids are encoded by more than one codon; the third nucleotide (wobble base) can often vary without changing the amino acid. Some amino acids have more than 3 codons

Why only one DNA strand is transcribed

For each protein-coding gene, only one strand (the coding strand) is transcribed into RNA; the other (template) strand is used as the template for complementary base pairing

Relationship between coding strand and mRNA

The mRNA sequence will more closely match the sequence of the coding strand (except T in DNA is replaced by U in RNA)

RNA vs DNA nucleotide difference

RNA contains uracil (U) instead of thymine (T); on paper, replace every T in DNA with U to convert to RNA sequence

Promoter function in transcription

Transcription factors bind to the TATA box and promoter region to recruit RNA polymerase and initiate transcription; NOT involved in translation or splicing

Transcription termination

Transcription ends when RNA polymerase encounters a terminator sequence; NOT when it encounters a stop codon (stop codons signal termination of translation, not transcription)

Eukaryotic gene structure summary

Promoter (with TATA box) upstream of transcription start site; cis-control elements/enhancers; exons and introns; 5'UTR and 3'UTR; poly(A) site at the end of the last exon

Effect of adding nucleotide to an intron

Will NOT disrupt amino acid sequence unless a splice site is affected; introns are removed from pre-RNA before translation

Exon nucleotide count

Each exon does NOT necessarily contain a multiple of 3 nucleotides; exons can be any length, but the combination of exons that are spliced together must maintain the reading frame

Determining UTR boundaries

Scientists distinguish UTR boundaries using experimental methods such as: RACE (Rapid Amplification of cDNA Ends), RNA-seq, and comparison of cDNA sequences to genomic DNA to identify transcribed but untranslated regions

Deletion of exon 1 vs exon 3 phenotype

Deleting exon 1 likely has a stronger phenotype because exon 1 contains the start codon (ATG) and deleting it prevents translation entirely; exon 3 deletion may preserve partial function if reading frame is maintained

Number of reading frames for a DNA sequence

6 possible reading frames (3 forward frames and 3 reverse complement frames); important when analyzing unknown sequences where coding strand is not known

PCR product reading frames

When the coding strand is unknown, all 6 reading frames must be considered because the sequence could be from the coding strand or the non-coding/template strand