Lecture #6 Flashcards
The Central Dogma
DNA is transcribed to make RNA
-Re-write the genetic information
RNA is translated to make protein
- Going from one language to another.
The Central Dogma: Gene Expression
Gene expression:
Information in a DNA sequence is translated into a product that has an effect on the cell/organism
Regulated by the cell
- Time (cell cycle)
- Amount (cell type)
Each gene is individually regulated
Transcription: An Overview
The number of RNA copies made from each gene varies
- few copies vs. hundreds of copies
The number of protein molecules made from each RNA strand varies
- A few copies vs. hundreds of copies
The longer an RNA strand remains functional, the more protein molecules are generated
- Half life
Similarities and Differences between DNA and RNA
Similar to DNA
- Chemical make-up
Polymer of nucleotides held together by phosphodiester bonds.
- 5`→3` polarity
- Bases added in a 5`→3` direction
- Similar base-pairing
- Uracil used instead of thymine
Different than DNA
- RNA uses a ribose sugar
- RNA uses the uracil instead of thymine
- The methyl group makes the base more stable.
- RNA is single stranded
The Structure and Function of RNA
Characteristics
- More flexible than DNA double helix
- Acts similarly to a polypeptide.
- Uses convention and non-conventional base-pairing methods
- Can form many different shapes
2d and 3d structures
Functions
- Transfers information
- Provides structural support
- Catalyzes reactions
Transcription vs. Replication Similarities
Similarities to DNA replication
- DNA opened unwound
- One strand acts as a template for synthesis of an RNA strand
- Ribonucleotides added in a 5→3 direction
Base pair complimentary rule
RNA chain is produced
Transcription vs. Replication Differences
Differences between replication and transcription
- RNA strand dissociates from the DNA strand after base pair addition
Another strand may be started before the first one is complete.
- RNA strand much shorter than DNA
- Different polymerases
RNA Polymerase vs. DNA Polymerase
Similarities - Catalyzes formation of the phosphodiester bond - 5' --> 3' direction Differences - RNAP carries out transcription - RNAP adds ribonucleotides - RNAP acts as its own helicase - RNAP does not require a primer - RNAP is less accurate
Products of Transcription
Messenger RNA (mRNA) – proteins
Non-messenger RNAs rRNA: ribosomal RNA (translation) tRNA: transfer RNA (translation) snRNA: small nuclear RNA (splicing) miRNA: microRNA (gene expression) siRNA: small interfering RNA (gene expression) Others…
Basic Structural Elements of Transcription
Promoter: DNA sequence - Required
- Signals the starting point for RNA synthesis
- Asymmetrical sequence – RNAP binds in one orientation
Top or Bottom strand can act as template.
- contains transcription start site (TSS) template.
TATA Box – can be within the promoter
5’-TATAAA-3‘
~ 25 bp upstream of the Transcription start site
- Binding site for TBP (TATA binding protein)
Transcription Terminator site
Bacterial Transcription
RNAP associates with DNA
- Slides along DNA
Recognizes promoter & binds more tightly
- Sigma factor recognizes promoter.
First 10 nucleotides are joined
RNAP exits promoter
- Lose the sigma factor.
RNA strand is elongated
RNAP reaches terminator signal and dissociates
Bacterial Transcription & Translation are Coupled
Bacterial DNA is not contained in a nucleus
Ribosomes bind newly transcribed mRNA and begin translation
Eukaryotic Transcription
Very Complex
- Genes are closer together in bacteria
- Bacteria DNA is not packaged into nucleosomes.
Three types of RNAP
- I and III responsible for tRNAs, rRNAs, and other small RNAs
Eukaryotic Transcription Continued
Requires general transcription factors during initiation
- Transcription Initiation Complex forms on promoters
Positions RNAP II
TFIID interacts with the promoter
- Binds TATA box through its TBP domain
- Distorts the DNA
- Recruits other complex subunits
Transcription in Eukaryotes
Other Transcription Factors interact with DNA and RNAP II
- TFIIF, TFIIE, TFIIH
TFIIH phosphorylates the RNAP II tail
- TFIIH has a kinase domain
- RNAP II dissociates from Transcription Initiation complex
RNAP II uses ribonucleotides to start and elongate the mRNA strand
RNAP II reaches terminator signal and dissociates
mRNA Splicing Part I
In eukaryotic genes, expressed sequences are interrupted by intervening sequences that are not expressed
- Exons: expressed sequences
- Intros: Intervening sequences
Both exons and introns are transcribed but both are not translated
What happens?
mRNA Splicing Part II
Intron sequences are removed by splicing
- Introns are “cut out” and the exons are “stitched” back together
Splice sites are recognized by short sequences of DNA
- Similar in all introns
- Located at both ends of the intron
mRNA Splicing Part III
Mediated by the spliceosome
- RNA and proteins
snRNAs (small nuclear RNAs)
snRNPs: snRNAs + proteins
General steps
- Recognition of branch point adenine
- 5
and 3splice sites are recognized - The branch point attacks the 5` splice site and forms a lariat
- 5` splice site is cleaved
- 3` splice site is cleaved
- 3
end of exon1 joins to 5end of exon2
The Advantages of mRNA Splicing
Alternative splicing
- Different splice sites are recognized
Different protein isoforms are possible
- Similar to full-length but missing specific features
- Used in many types of mammalian cells
- Can be cell specific
Eukaryotic Transcription & Translation are Separate
Eukaryotic DNA is contained in the nucleus
Transcription machinery is in the nucleus
Translation machinery is in the cytoplasm
Question: How does the mRNA get to the cytoplasm?
Answer: The Nuclear Pore Complex
mRNAs are post-transcriptionally modified
Purpose
- Protection from degradation before export
- Provides signals to the nuclear pore complex for export
- Identification of RNA strand as mRNA
5` capping
- Capping factors bind the tail of RNAP II
- Attaches a methylated guanine nucleotide to the 5` end of the mRNA
- Occurs during transcription
- Signals nuclear pore complex
mRNAs are post-transcriptionally modified continued
3` Polyadenylation Signal
- 3` end of mRNA is cleaved and trimmed – sequence specific
- Adenine nucleotides are added to the 3` end (100-250nt)
- Protects the 3` end from degradation (stability)
- Signals nuclear pore complex to export
- Occurs during or after mRNA splicing
Transport of mature mRNA into the cytoplasm
Mature mRNA is recognized by the Nuclear Pore Complex
- Aqueous channels within the nuclear membrane
- Connect the nucleoplasm with the cytoplasm
Signals recognized by Nuclear Pore Complex
- 5’ cap
- 3’ polyadenylation
- Proteins that mark completion of splicing.
