27 28 105 fa13 transcription and translation skel

Transcription and Translation

BIOL 105

Dr. Corl

October 25, 2013

The Central Dogma

• DNA codes for RNA, which codes for protein.

The Central Dogma

• Transcription is a process in which:– The sequence of bases in a particular stretch of

DNA (a gene) specifies the sequence of bases in an mRNA molecule.

• Translation is a process in which:– A particular mRNA molecule then specifies the

exact sequence of amino acids in a protein.

• Thus, genes ultimately code for proteins.

Transcription in Bacteria

• In transcription:– Instructions stored in DNA are “transcribed”

through the synthesis of an mRNA transcript.

• Transcription performed by RNA polymerase.

• Three phases of transcription:– Initiation, elongation, and termination.

Transcription in Bacteria

• mRNA transcript is synthesized by RNA polymerase in the 5’ to 3’ direction.

• Template strand:– DNA strand that is read (transcribed) by RNA polymerase.

• Non-template strand:– DNA strand that is not read by RNA polymerase.

Transcription in Bacteria

• “Activated” complementary ribonucleotide monomers are added on via condensation reactions, resulting in phosphodiester bonds.

Bacterial RNA Polymerase

• A holoenzyme:– An enzyme made up of a core enzyme and other

required proteins.– Bacterial RNA polymerase is made up of a core

enzyme and a regulatory subunit, sigma.

Bacterial RNA Polymerase

• Core enzyme:– Contains the active site where mRNA is synthesized.

• Sigma:– A regulatory factor required for initiation of transcription.– Tells core enzyme where in a DNA sequence to start transcription.

Transcription: Initiation

• Transcription initiation begins at specific sequences of DNA called promoters.

• Two important bacterial promoter regions :– Named the -10 box and the -35 box.– Sigma recognizes these promoter regions and

brings the RNA polymerase core enzyme to the promoter to initiate transcription.

Transcription: Initiation

• 1.) sigma binds to specific promoter regions of DNA (-35 box and -10 box).

Core enzyme

Transcription: Initiation

• 2.) DNA double helix is opened and the template strand of DNA is threaded through RNA polymerase core enzyme active site. mRNA synthesis begins.

Transcription: Initiation

• 3.) Initiation is complete. Sigma dissociates from core enzyme. mRNA synthesis (transcription) continues.

Transcription: Elongation

• Core enzymes of RNA polymerase moves along the DNA template and continues to catalyze the addition of complementary ribonucleotides to the growing mRNA transcript.

Transcription: Termination

• RNA polymerase encounters a termination signal within the DNA template, which codes for RNA forming a hairpin loop structure.

• Hairpin causes RNA polymerase to separate from RNA transcript, ending transcription.

Bacterial Transcription: Summary

• Initiation:– Sigma brings RNA polymerase holoenzyme to

promoter region of DNA.– DNA helix is opened and transcription begins.– Sigma releases and transcription continues.

• Elongation:– Complementary ribonucleotides are added to the

growing mRNA transcript as specified by the DNA template strand.

• Termination:– RNA polymerase reaches a termination signal in

the DNA template.– mRNA forms a hairpin loop.– mRNA dissociates from RNA polymerase.

Transcription in Eukaryotes

• Overall similar to bacterial transcription.

• Some differences include:– Basal transcription factors:

• Proteins that bind DNA promoters independant of RNA polymerase.

• RNA polymerase then binds to basal transcription factors and transcription begins.

– Greater diversity and complexity of promoters:• Many promoters include a TATA box sequence.

Transcription in Eukaryotes

• Three types of RNA polymerase:

– RNA polymerase II: • Catalyzes transcription of genes that code for

proteins, forming mRNA.

– RNA polymerase I and RNA polymerase III:• Catalyze transcription of non-protein coding

genes (e.g. genes coding for ribosomal rRNAs and genes coding for transfer tRNAs).

Eukaryotic mRNA Processing

• After transcription but before translation, specific regions of the primary RNA transcript are spliced out (cut out) and degraded during RNA processing.

• The term intron can be used in two ways:– A stretch of RNA that does get spliced out and will NOT

be a part of the final mature spliced RNA transcript.– A stretch of DNA that codes for an RNA intron.

Exon 1 Exon 2 Exon 3Intron 1 Intron 2

Eukaryotic mRNA Processing

• After transcription but before translation, specific regions of the primary RNA transcript are spliced out (cut out) and degraded during RNA processing.

• The term exon can be used in two ways:– A stretch of RNA that does NOT get spliced out and will

be a part of the final mature spliced RNA transcript.– A stretch of DNA that codes for an RNA exon.

Exon 1 Exon 2 Exon 3Intron 1 Intron 2

Eukaryotic mRNA Processing

-Intron regions of the primary mRNA transcript are spliced out by small nuclear ribonucleoproteins (snRNPs), which assemble to form a spliceosome (a ribozyme).

Eukaryotic mRNA Processing

• Intron regions of the primary mRNA transcript are spliced out by snRNPs, which assemble to form a spliceosome. (occurs within nucleus)

Eukaryotic mRNA Processing

• Additionally, a 5’ cap is added to the very 5’ end of the mRNA transcript:

– 5’ cap serves as recognition signal for the translation machinery of the cell.

Eukaryotic mRNA Processing

• Finally, a poly (A) tail is added to the very 3’ end of the mRNA transcript:

– Extends the life of the mRNA by protecting it from enzymatic degradation.

Transcription: Summary

• Note the similarities and differences between bacterial vs. eukaryotic transcription!

• Eukaryotic transcription tends to be more complex.

Translation

• In translation:– The sequence of bases in mRNA is

converted (“translated”) to an amino acid sequence of a protein.

• Ribosomes catalyze the translation of mRNA sequence into protein.

Translation in Bacteria

• In bacteria, transcription and translation can occur simultaneously:– Ribosomes begin translating an mRNA even before RNA polymerase

has finished synthesizing it!

Translation in Bacteria

• In bacteria, transcription and translation can occur simultaneously:– Ribosomes begin translating an mRNA even before

RNA polymerase has finished synthesizing it!

Translation in Eukaryotes

• Transcription and translation are separated:– Transcription takes place in the nucleus.– Translation takes place in the cytoplasm.

Translation

• How do mRNA codons interact with amino acids?

Translation

• Adapter molecules:– Small RNAs called transfer RNA (tRNA)– Hold amino acids in place and interact directly and

specifically with a codon in mRNA.

Transfer RNA (tRNA)

• Different tRNAs covalently link to specific amino acids, forming aminoacyl tRNAs.

• Aminoacyl tRNA synthesizes:– Enzymes which catalyze the addition of

amino acids to tRNAs.

Loading Amino Acid Onto tRNA

• Amino acid binds to its specific aminoacyl tRNA sythnetase, which then covelantly attaches the amino acid onto a specific tRNA.

Step 1

Step 2

Step 3

Step 4

Loading Amino Acid Onto tRNA

• Is ATP expended in this process? Yes!• Aminoacyl tRNA = “CHARGED” tRNA

Step 1

Step 2

Step 3

Step 4

Aminoacyl tRNA

• Aminoacyl tRNAs then travel to ribosomes and transfer amino acids to growing polypeptides.

• Each tRNA carries a specific amino acid that can be transferred to protein.

Transfer RNA Structure

• tRNA secondary structure resembles a “cloverleaf.”

tRNA Secondary Structure

• 3’ end of tRNA: binding site for amino acids.• Triplet loop at opposite end (anticodon):

– Interacts with complementary codon on mRNA.

tRNA Tertiary Structure

• tRNA has an “L-shaped” tertiary structure.• Each tRNA has a distinct anticodon and attached

amino acid.

Wobble Hypothesis

• There are 61 different codons but only 40 different tRNAs.

• Explained by “wobble hypothesis”:– The anticodon of certain tRNAs can bind

successfully to a codon whose third position requires nonstandard base pairing.

– Allows one tRNA to be able to base pair with more than one type of codon.

Ribosomes

• Composed of both ribosomal protein and ribosomal RNA (rRNA).

• Can be separated into two subunits:– Large subunit and small subunit.

• There are three sites within a ribosome that tRNAs can reside: – The A site, the P site, and the E site.

Ribosome: Structure

• Large subunit and small subunit.• Three sites where tRNAs can reside: A, P and E.

Large subunit

Small subunit

E P A

Ribosome: Structure and Function

• Each tRNA binds at its anticodon to its corresponding mRNA codon and transfers its amino acid to a growing polypeptide chain.

Phases of Protein Synthesis• initiation:

– Ribosomal subunits and mRNA assemble.– Translation begins at the AUG start codon.

• elongation:– Amino acids are transferred one by one from aminoacyl

tRNAs to a growing polypeptide chain.

• termination:– Stop codon in mRNA causes protein to be released from

ribosome.– Ribosomal subunits separate from mRNA and from one

another.

Initiation: Step 1

• mRNA is targeted to the ribosome:– Ribosome binding site on mRNA binds to

complementary sequence on small subunit of ribosome, with the help of proteins called initiation factors.

Initiation: Step 2

• Translation begins at the AUG start codon:– Initiator aminoacyl tRNA, bringing in the first amino acid,

binds to the mRNA’s start codon, AUG.

Initiation: Step 3

• LARGE subunit of ribosome binds, placing initiator aminoacyl tRNA in the P-site.

Elongation: Step 1

• Incoming aminoacyl tRNA binds to the codon in theA site via complementary base pairing between anticodon and codon.

Elongation: Step 2

• The amino acid in the P site:– Breaks the bond with its tRNA.– Forms a peptide bond with the amino acid in the A site.

Elongation: Step 3

• Translocation:– Ribosome moves down to the next codon on mRNA.– Results in tRNAs being shifted over one spot:

• tRNA in P site moves to E site.• tRNA in A site moves to P site.

Another Elongation Cycle

• 1.) Incoming aminoacyl tRNA enters into the __ site.• 2.) peptide bond formation.

– rRNA acts as a catalyst: ribozyme

• 3.) Translocation.

• multiple ribosomes assembled along a single mRNA, synthesizing proteins from the same mRNA at the same time.

Polyribosome

Termination: Step 1

• Occurs when a stop codon is revealed in the A site.• A protein called a release factor enters the A site

and promotes the hydrolysis of the bond linking the tRNA in the P-site with its polypeptide.

Termination: Steps 2 and 3

• Hydrolysis reaction (cleavage of bond between tRNA and polypeptide) frees the polypeptide

• Ribosomal subunits, mRNA, tRNAs, and polypeptide all _________ from one another.

Post-Translational Modifications

• Many proteins are modified after translation is complete:

– Many proteins fold with the help of molecular chaperones.

– Proteins can be modified by attachment of sugars (glycosylation) in the rough ER and/or Golgi.

– Proteins can be phosphorylated or dephosphorylated, modifying their activity.

Mutations

• Mutation:

– Any permanent change in an organism’s DNA.

– Changes the genotype (DNA) of the cell.• This may then result in a change in mRNA transcript

sequence, resulting in:– Change in sequence of amino acids of the translated protein.

Point Mutations

• A single base pair change, often as a result of errors in DNA replication.

• Can affect the primary structure of the polypeptide that is ultimately translated.

Point Mutations

• missense mutation:– Point mutation in a gene’s DNA sequence that

ultimately results in a single amino acid change in the protein encoded by that gene.

– Can often be deleterious:• Reduces the individual’s fitness.

Point Mutations

• silent mutation:– Does not change amino acid sequence.

• nonsense mutation:– Results in an early stop codon: shortened protein.

• frameshift mutation:– Addition or deletion of a nucleotide causes entire reading

frame to be shifted.

Chromosome-Level Mutations

• Chromosome INVERSION and translocation:– Can result in altered patterns of gene expression.

Summary• Transcription:

– Bacterial transcription– Eukaryotic transcription

• Translation:– Ribosomes– tRNA and aminoacyl tRNA– Ribosomal protein synthesis

• Mutations:– Point mutations– Chromosomal mutations

Review Questions on Transcription

• Contrast the functions of sigma versus the core enzyme of bacterial RNA polymerase.

• What events occur during transcription initiation? Elongation? Termination?

• How does eukaryotic transcription differ from bacterial transcription?

• What types of post-transcriptional processing occur in eukaryotes? What is splicing?

Review Questions on Translation

• What is the function of tRNA? mRNA? rRNA?

• What events occur during the three major phases of translation?

• What are the A, P, and E sites of a ribosome?

• What are the differences between silent, missense, nonsense, and frameshift mutations?