Transcription and RNA Processing

Central Dogma

DNA is transcribed into RNA and RNA is translated into protein.

Transcription

 A portion of a DNA nucleotide sequence (a gene) is copied into an RNA nucleotide sequence

Differences between DNA and RNA

• The nucleotides in RNA are ribonucleotides -> they contain the sugar ribose instead of deoxyribose

• RNA has uracil instead of thymine as a nitrogenous base

• RNA is single-stranded while DNA is double-stranded

  • RNA can fold into 3D shapes -> allows it to have specific structural and catalytic functions

• DNA strands are hydrogen-bonded

• RNA strands are shorter than DNA strands

Where do transcription and translation take place in the cell

Transcription is in the nucleus, and translation is in the cytoplasmic ribosomes

mRNAs

messenger RNAs -> code for proteins

rRNASs

•  ribosomal RNAs

• for the basic structure of the ribosome and catalyze protein synthesis

tRNAs

• transfer RNAs

• central to protein synthesis as adapters between mRNA and amino acids 

Telomerase RNA

 serves as the template for the telomerase enzyme that extends the ends of chromosomes

snRNAs

• small nuclear RNAs

• function in variety of nuclear processes i.e. splicing of pre-mRNA

snoRNAs

small nucleolar RNAs, help to process and chemically modify rRNAs

lncRNAs

long non coding RNAs

not all of them have a function; some serve as scaffolds and regulate diverse cell processes, including X-chromosome inactivation

miRNAs

MircroRNAs, regulate gene expression by blocking translation fo specific mRNAs and causing their degradation 

siRNAs

small interfering RNAs

turn of gene expression by blocking transition of specific mRNAs and causing their degradation
can also regulate RNA stability

non-coding RNAs

final product is RNA and not protein

RNA Polymerase II transcribes all protein -coding genes, as well as snoRNA genes, miRNA genes, siRNA genes, lncRNA genes, and most snRNA genes.

Which RNA polymerase transcribes all protein-coding genes?

RNA polymerase reads the DNA template strand in the 3’->5' direction, while synthesizing RNA in the 5’ -> 3 ’ direction

• RNA is complementary and antiparallel to the DNA template strand.

What is the direction of transcription (RNA synthesis)

Base pairing for RNA synthesis (transcription)

• DNA → RNA base pairing:

  • A → U

  • T → A

  • C → G

  • G → C

5′–AUG CCU GAA–3′

Write the RNA transcript made from this DNA template:
3′–TAC GGA CTT–5′

Promoter

•  upstream 5’ end; where transcription starts

• RNA polymerase binds to this DNA nucleotide sequence to start transcription

• recruits the transcription machinery and specify where transcription begins

Enhancers

• noncoding regulatory regions that contain clusters of DNA-binding sites for transcription factors

• When they are bound, they increase the likelihood of transcription initiation from a nearby promoter

UTR (untranslated region)

 non-coding regions at the 5’ and 3’ ends of the coding sequence that regulate transcription, processing, stability, and translation

5’ UTR (leader sequence)

segment of mRNA from the transcription start site up to the start codon (AUG)

3’ UTR (trailer sequence)

the segment after  the stop codon, often containing regulatory elements for mRNA stability, localization, and degradation

Exon

 intervening coding regions

To mature RNAs, introns must be removed, and exons must be added together (RNA splicing)

intron

 intervening non-coding regions

• Promoter: DNA sequence where transcription begins; includes the TATA box in many genes.

• RNA polymerase II: Enzyme that synthesizes mRNA from the DNA template.

• Transcription factors: Proteins that help RNA polymerase bind to the promoter and initiate transcription.

• TATA-binding protein (TBP): Recognizes and binds the TATA box.

• DNA template strand: Strand read by RNA polymerase (3′ → 5′).

• Terminator: DNA sequence that signals the end of transcription (termination mechanisms vary in eukaryotes

What are the key components involved in transcription, including proteins and DNA sequences?

Transcription factors

• Proteins that help RNA polymerase bind to the promoter and initiate transcription.
  
  

• General - present in all cell types

• Regulatory - present in specific cell types

  • Bind DNA and and interact with mediator

• Enhanceosome - multiple transcription factors working together

  • Enhances binding to DNA and stabilizes transcriptional complex --

• Activators - Transcription factors bound to enhancer sequences that promote formation of transcriptional complex

• Repressors - transcription factors bound to silencer sequences that inhibit the formation of transcriptional complex

TATA box

Sequence in the promoter region of many eukaryotic genes that binds a general transcription factor (TFIID) and hence specifies the position at which transcription is initiated

for high regulated genes → actively controlled between no transcription and high levels of transcription, depending on the time/stage/environmental influence and cell types.

General transcription factors

present in all cells (TFIIA, TFIIB, etc). They help to position RNA Pol II at the promoter

Regulatory transcription factors

• cell-type specific

• help make the decision (and efficacy) in transcription

• RNA is processed while it is still being synthesized by RNA polymerase

• 5’ capping, early splicing, and 3’ end formation all happen while the pre-mRNA is still being synthesized by RNA polymerase II, not only transcription

Why is RNA processing considered a co-transcriptional process instead of a post-transcriptional process

TFIIH phosphorylates CTD (C-terminal domain) polypeptide tail of RNA Pol II, causing the polymerase to be released from the general factors and begin the elongation phase of transcription

What modifications on RNA poly II alllow RNA processing to be co-transcriptional

• processing enzymes for splicing and 3’ end processing

  • additional phosphates are added to the rna polymerase tal, which attracts splicing and 3’ end processing enzymes

What other processing si recruited by RNA Pol III altered CTD

What are the three types of RNA processing

1. 5’ Capping

2. 3’ Polyadenylation (Poly A Tail Addition)

3. RNA Splicing

5’ Capping

1. Protects the transcript from degradation 

2. Helps the ribosome attach to the transcript in translation

3’ Polyadenylation (Poly A Tail Addition)

• Enzyme PAP (poly A Polymerase) adds 200 A’s, one at a time. ATP required, 5’-3’ covalent bonds link nucleotides (RNA synthesis): does not require a template.|
  
  Makes transcript more stable (protects from degradation)

• Assists in the export of mRNA (from the nucleus into the cytosol)

• 
  

RNA Splicing

Eukaryotic genes are broken up into small pieces of coding sequences (exons) interspersed with longer non-coding sequences (introns) 

• On average, 11 introns in each gene.

• Each splicing event occurs between one 5’ splicing site and one 3’splicing site.

• Proceeds through two sequential phosphoryl-transfer reactions.

• Lariat -> excised intron

• Splicing machinery recognizes the 5’ splice site (splicing donor), 3’ splice site (splicing acceptor), and branch point in the intron sequence that forms the base of the lariat.

Mechanism for RNA Splicing

5′ cap and 3′ poly(A) tail shield mRNA from exonucleases, block end‑to‑end degradation, and recruit stabilizing proteins, greatly extending mRNA half‑life 

How does 5’capping and 3’ polyadenylation protects mRNA from degradation? 

Transcript of a given gene can be spliced in more than one way (alternative splicing) so the same gene can produce different but related proteins

What is the benefit of having introns and then splice them off?

snRNAs (small nuclear RNAs): recognize binding sites

snRNAs + protein=snRNP (ribonucleoprotein)

snRNP is the core of the spliceosome.

snRNAs base pair with pre-mRNA.

What are the major components of the spliceosome? Describe the general steps of splicing (includes the formation of a lariat).

 

Abnormal RNA splicing, potentially resulting in intron retention or exon skipping

What is the result of a mutation disrupting a conserved splice donor site at exon-intron boundary?

Are MAPKs a family of tyrosine kinases?

no, they are serine/thereonine kinases

Ras activates Raf (MAPKKK), which phosphorylates MEK (MAPKK), which phosphorylates ERK (MAPK). Each kinase activates the next.
Ras → MAPKKK (Raf) → MAPKK (MEK1/2) → MAPK (ERK1/2)

What is the order of the core MAPK cascade after Ras?

It phosphorylates ERK on both a threonine and a tyrosine

MEK phosphorylates ERK on a threonine and a tyrosine in its activation loop, which activates ERK.

What makes MEK a dual-specificity kinase?

Akt is recruited to PI(3,4,5)P3 and activated, then active Akt detaches from PI(3,4,5)P3

FRET rises when Akt is at the membrane bound to PIP3, close to the donor. Once Akt is phosphorylated and activated, it dissociates from the membrane, so FRET drops.

In the FRET experiment (CFP on PI(3,4,5)P3, YFP on Akt), FRET rises within minutes of adding growth factor, then drops. Why?

The two tagged molecules have moved very close together

FRET only occurs when the donor and acceptor are very close, so higher FRET means closer proximity, here Akt binding PI(3,4,5)P3 at the membrane.

What does a rise in FRET between CFP and YFP tell you?

Initiation of transcription of RNA polymerase II

Only for transcription of genes with TATA box promoter. TFIID subunit binds to TATA box, leading to bending of DNA, additional general factors and RNA Pol II recruited to the initiation complex, including TFIIH which unwind DNA, transcription starts.

Transcription initiation:

Transcriptional activators that bind to DNA enhancer sequences Mediation complex - bridges transcriptional activator, general transcription factors, and RNA Pol II 3. Chromatin modifying enzymes

Mediators

• Large protein complexes that bridge the activator that is bound to the enhancer, with RNA pol II that is bound to the promoter

• Facilitates assembly of the RNA Pol II preinitiation complex

• Histone acetylase activity - maintain a promoter region in hyperacetylated state

Cis regulatory elements

DNA loops to bring all of the elements together - Regulating same DNA sequence that they are bound to