Unit 10

• Gene expression: gene function either at molecular level or at level of traits

• Protein-coding genes: genes that code polypeptides

• Non-coding RNAs (ncRNA): RNAs that don’t code polypeptides

10.1 Overview of Gene Expression

• General steps of gene expression follows

  • Transcription: produces an RNA copy of a DNA strand

    • Protein-coding genes are used to make an RNA molecule that can specify a polypeptide with a particular amino acid sequence

      • RNA is known as mRNA

  • Translation: the process of synthesizing a specific polypeptide

    • a sequence of bases in mRNA are “translated” into an amino acid sequence of a polypeptide

• Transcription of DNA to mRNA and translation of mRNA to polypeptide constitutes a central dogma of gene expression to a molecular level

  • Central dogma: refers to steps of gene expression

• Central dogma applies to bacteria, archaea, and eukaryotes

  • In eukaryotes there is an additional step between translation and transcription

• RNA modification: a change in structure of an RNA molecule

  • modified to make it functionally active mRNA

• In bacteria, transcription/translation occurs in cytoplasm

• In eukaryotes, transcription occurs in nucleus, translation occurs in the cytosol

Protein products of genes largely determine an organism’s characteristics

• Genes contain info necessary to form an organism

  • genes help code proteins that aid in this

10.2 Transcription

• Allows for information in DNA to be accessed without damaging the DNA

• Two types of mRNA used during transcription

  • Transfer RNA (tRNA): carries the amino acids needed to make the protein

    • carries an anticodon that is complementary to the codon on a mRNA

  • Ribosomal RNA (rRNA): forms part of the ribosome, where translation occurs

During Transcription, RNA polymerase use a RNA template to make RNA

• Transcription occurs in 3 steps: initiation, elongation, termination

  • Initiation: Recognition step

    • in bacteria, protein called sigma factor binds to RNA polymerase

      • sigma factor recognizes base factor, binds there, allowing RNA polymerase to bind to promoter

    • Initiation is completed when DNA strands form an open complex around 10-15 bp long

  • Elongation: RNA polymerase synthesizes RNA transcript

    • Sigma factor is released, RNA polymerase slides along DNA maintaining open complex

    • DNA strand that is used as a template is known as template strand

    • For protein coding genes, the opposite DNA strand is called the coding strand

      • coding strand has same sequence of bases as the resulting mRNA

  • Elongation II: Nucleotides bind to template strand and are covalently connected in the 5’ to 3’ direction

    • as it is transcript, DNA complex turns back to double helix

  • Termination: RNA polymerase reaches a terminator, causing newly made RNA transcript to dissociate from the DNA

Transcription in Eukaryotes involves more proteins

• Basic features of transcription are similar among all organisms

  • all genes are have promoters

  • Transcription in eukaryotes involve a greater number of protein components

• RNA polymerase II requires five transcription factors to initiate transcription

  • transcription factors: proteins that influence RNA polymerase ability to transcribe genes

10.3 RNA Modifications in Eukaryotes

• pre-mRNAs undergo modifications before they are mature mRNAs

• Eukaryotic pre-mRNAs having coding sequences that are separated are later removed

  • sections that are not translated are called introns

  • sequences contained in mRNA are called exons

• RNA splicing: the process of pre-mRNA undergoing modifications to become mature mRNA

  • introns are removed and exons are connected

• After modifications, mature mRNA leave nucleus and go to cytosol

End of Eukaryotic Pre-mRNAs are modified by addition of 5’ cap and 3’ Poly-A tail

• Mature mRNAs of eukaryotes have a modified guanine attached to 5’ end, event known as capping

  • 5’ cap is recognized by cap-finding proteins needed for proper exit of mRNAs from nucleus

  • cap prevents degradation while mRNA is in cytosol

  • cap structure allows mRNA to bind to a ribosome for translation

• 3’ end has a string of adenine nucleotides, typically 100 - 200 nucleotides in length, known as poly A tail

  • not coded in gene sequence

  • aids in export of mRNA from nucleus

Splicing involves removal of introns and linkage of exons

• Splicing is rare in unicellular eukaryotes, but common in multicellular eukaryotes

• introns are removed from pre-mRNA by a complex called spliceosome

  • spliceosome composed of several subunits called snRNPs (snurps)

• Splicing occurs in 4 steps

  • spliceosome subunits bind to specific sequences at three locations in the intron RNA

    • one is termed branch site, and two sequences at intron-exon boundaries are called 5’ splice site and 3’ splice site

  • Binding causes intron to loop outward, bringing the two exons together

  • 5’ splice site is cut, 5’ end of intron becomes covalently attached to branch site

  • 3’ splice site is cut, 2 exons are covalently attached to each other

    • intron, still in a loop, is released and degraded

• Ribozyme: RNA molecules that catalyzes a chemical reaction

• Alternative splicing: allows a single gene to code two or more polypeptides with differences in their amino acid sequences, increasing protein diversity

• Sometimes introns occur in RNA and tRNA molecules, removed not by spliceosome

  • instead it does self-splicing, it catalyzes to remove introns

10.4 Translation and the Genetic Code

• RNA relies on the genetic code in order to be translated into amino acid sequence

• Code is read via codons

  • a group of three nucleotides

• Genetic code consist of 64 different codons (table is in lab manual)

  • genetic code is nearly universal

• Genetic code is said to be degenerate/redundant

  • more than one codon can specify the same amino acid

During Translation, mRNA is used to male polypeptides with specific amino acid sequence

• Ribosomal-binding site: a site that is located near 5’ end of an mRNA provides location for ribosome to bind to mRNA

• Start codon: first codon used in translation; determines where translation begins, may be removed from polypeptide

• Coding sequence: region that begins with start codon and specifies the entire amino acid sequence of a polypeptide; it consists of many codons

• Stop codon: the last codon, which signals the end of translation

• start codon defines reading frame of mRNA

  • beginning at start codon, each adjacent codon is read in 3 bases

    • called triplet, read from 5’ to 3’

DNA stores information, mRNA and rRNA accesses information

• recognition between nucleotide sequences of mRNA and tRNA is necessary

  • tRNA functions as a translator between mRNA codon and amino acid

• Anticodon: 3 base sequence in a tRNA molecule that is complementary is a codon in mRNA

  • this allows anticodon in tRNA to bind to codon in mRNA

• Direction of polypeptide synthesis parallel 5’ to 3’ orientation of mRNA

  • First amino acid is said to be at amino end (N-terminus) of a polypeptide

    • refers to presence of nitrogen at the end

  • Peptide bonds connect amino acids together

    • forms between carboxyl group

  • Last amino acid in a completed polypeptide doesn’t have amino acid attach to its carboxyl group

    • said to be located at carboxyl end/C terminus

Synthetic RNA helped researchers decipher the genetic code

• In vitro translation systems: mixture of components isolated from cells that can translate mRNA into polypeptides

10.5 The machinery of translation

• tRNAs of all species share common features

  • 2D structure of tRNA resembles a cloverleaf

    • structure has a 3 stem loops and a fourth stem with a 3’ single stranded region

  • Anticodon in the middle loop

  • 3’ single-stranded regions is the amino acid attachment site

  • 3D structure of tRNA molecules involve additional finding of secondary structure

Aminoacyl-tRNA synthetases charge tRNAs

• tRNA must have appropriate amino acid attach to 3’ end

• aminoacyl-tRNA synthetases: enzymes that catalyze the attachment of amino acids to tRNA molecules

• Occurs in 4 steps

  • a specific amino acid and ATP bind to the enzyme

  • amino acid is activated by covalent attachment of an adenosine monophosphate (AMp) and pyrophosphate (PPi) is released

  • a specific tRNA binds to enzyme; AMp is released

  • tRNA and attached amino acid is called charged tRNA, or an aminoacyl tRNA, is released from enzyme

Ribosomes are composed of rRNA and proteins

• Translation takes place in ribosomes

• Ribosomes are composed of large and small subunits

• Made from proteins and rRNAs

Components of Ribosomal subunits form functional sites for translation

• James Watson proposed 2 sites for tRNA binding to ribosome

  • Peptidyl Site (P site) and Aminoacyl site (A site)

• Later expanded to 3 site model

  • Exit site (E site)

10.6 The stages of translation

• Translation occurs in 3 stages

  • Initiation: one mRNA, first tRNA, and ribosomal subunits assemble into a complex

  • Elongation: Ribosome moves in the 5’ to 3’ direction from start codon in mRNA toward stop codon

  • Termination: ribosome reaches the stop codon, complex disassembles, releasing completed polypeptide

Translation is initiated with assembly of mRNA, tRNA, and ribosomal subunits

• during initiation, complex is formed between mRNA, first tRNA, and the ribosomal subunits

• Requires translation factors known as initiation factors

• 3 step process in bacteria

  • mRNA binds to small ribosomal units aided by ribosomal-binding site near 5’ end of mRNA

  • Specific tRNA, the initiator tRNA, recognizes start of codon and binds to it

  • Large ribosomal subunit associates with small subunit, indicating end of initiation stage

    • initiator tRNA is located in P site of the ribosome

• Eukaryotic species differ in 3 ways

  • Eukaryotic mRNAs have a 7-methylguanosine cap (5’ cap) at their 5’ end of initiation stage

    • Cap is recognized by cap-binding proteins that promote the binding of mRNA to small ribosomal sub unit

  • The first AUG codon is not always the start codon

  • In eukaryotes, the initiator tRNA carries a methionine, not formyl-methionine

Polypeptide Synthesis occurs during elongation stage

• Involves covalent bonding of amino acids to each other to create polypeptide

• In order to elongate polypeptide, a tRNA brings a new amino acid to ribosome, and is attached to the end of growing polypeptide

• 3 steps

  • Binding of a charged tRNA: charged tRNA carry a single amino acid binds to A site

    • occurs because anticodon in tRNA is complementary to the codon in the mRNA

    • Hydrolysis of GTP by proteins that function as elongation factors provided energy for binding of tRNA to A site

  • Peptide bond formation and peptidyl transfer: peptide bond is formed between amino acid at A site and growing polypeptide

    • Peptidyl Transfer Reaction: polypeptide is removed from tRNA in P site and transferred to amino acid at A site

  • Translocation and exit of uncharged tRNA: ribosome moves toward 3’ end of mRNA by exactly one codon, shifting tRNAs on P and A sites to the E and P sites

    • uncharged tRNA exits E site

Termination and Stop Codons

• 3 stop codons: UAA, UAG, UGA

  • recognized by protein release factor

    • Release factor structure mimics tRNA, letting it fir into A site

• 3 steps to termination

  • Release factor binds to stop codon at A site

    • completed polypeptide is attached to tRNA as P site

  • Bond between polypeptide and tRNA is hydrolyzed, releasing polypeptide and tRNA from ribosome

  • mRNA, ribosomal subunits, and release factor dissociate

• Bacteria have 2 release factors, while eukaryotes have one